Interferon-alpha polypeptides and conjugates

ABSTRACT

The present invention provides interferon-alpha polypeptides and conjugates, and nucleic acids encoding the polypeptides. The invention also includes compositions comprising these polypeptides, conjugates, and nucleic acids; cells containing or expressing the polypeptides, conjugates, and nucleic acids; methods of making the polypeptides, conjugates, and nucleic acids; and methods of using the polypeptides, conjugates, and nucleic acids.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/848,827filed on May 19, 2004 (now U.S. Pat. No. 7,314,613), which is acontinuation-in-part of U.S. application Ser. No. 10/714,817 filed onNov. 17, 2003 (abandoned), which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/502,560 filed on Sep. 12, 2003 and U.S.Provisional Application Ser. No. 60/427,612 filed on Nov. 18, 2002, thedisclosures of each of which are incorporated by reference herein intheir entirety for all purposes.

COPYRIGHT NOTIFICATION

Pursuant to 37 C.F.R. 1.71(e), Applicants note that a portion of thisdisclosure contains material which is subject to copyright protection.The copyright owner has no objection to the facsimile reproduction byanyone of the patent document or patent disclosure, as it appears in thePatent and Trademark Office patent file or records, but otherwisereserves all copyright rights whatsoever.

FIELD OF THE INVENTION

The present invention relates generally to polynucleotides andpolypeptides encoded therefrom, conjugates of the polypeptides, as wellas vectors, cells, antibodies, and methods for using and producing thepolynucleotides, polypeptides, and conjugates.

BACKGROUND OF THE INVENTION

Interferon-alphas are members of the diverse helical-bundle superfamilyof cytokine genes (Sprang, S. R. et al. (1993) Curr. Opin. Struct. Biol.3:815-827). The human interferon-alphas are encoded by a family of over20 tandemly duplicated nonallelic genes and psuedogenes that share85-98% sequence identity at the amino acid level (Henco, K. et al.(1985) J. Mol. Biol. 185:227-260). Genes which express activeinterferon-alpha proteins have been grouped into 13 families accordingto genetic loci. Known expressed human interferon-alpha proteins andtheir allelic variations are tabulated in Allen G. and Diaz M. O. (1996)J. Interferon and Cytokine Res. 16:181-184.

Interferon-alphas have been shown to inhibit various types of cellularproliferation, and are especially useful for the treatment of a varietyof cellular proliferation disorders frequently associated with cancer,particularly hematologic malignancies such as leukemias. These proteinshave shown antiproliferative activity against multiple myeloma, chroniclymphocytic leukemia, low-grade lymphoma, Kaposi's sarcoma, chronicmyelogenous leukemia, renal-cell carcinoma, urinary bladder tumors andovarian cancers (Bonnem, E. M. et al. (1984) J. Biol. Response Modifiers3:580; Oldham, R. K. (1985) Hospital Practice 20:71).

Interferon-alphas are also useful against various types of viralinfections (Finter, N. B. et al. (1991) Drugs 42(5):749).Interferon-alphas have activity against human papillomavirus infection,Hepatitis B, and Hepatitis C infections (Finter, N. B. et al., 1991,supra; Kashima, H. et al. (1988) Laryngoscope 98:334; Dusheiko, G. M. etal. (1986) J. Hematology 3 (Supple. 2):S199; Davis, G L et al. (1989) N.England J. Med. 321:1501). The role of interferons and interferonreceptors in the pathogenesis of certain autoimmune and inflammatorydiseases has also been investigated (Benoit, P. et al. (1993) J.Immunol. 150(3):707).

Although these proteins possess therapeutic value in the treatment of anumber of diseases, they have not been optimized for use aspharmaceuticals. For example, dose-limiting toxicity, receptorcross-reactivity, and short serum half-lives significantly reduce theclinical utility of many of these cytokines (Dusheiko, G. (1997)Hepatology 26:112 S-121S; Vial, T. and Descotes, J. (1994) DrugExperience 10:115-150; Funke, I. et al. (1994) Ann. Hematol. 68:49-52;Schomburg, A. et al. (1993) J. Cancer Res. Clin. Oncol. 119:745-755).Diverse and severe side effect profiles which accompany interferonadministration include flu-like symptoms, fatigue, hallucination, fever,hepatic enzyme elevation, and leukopenia (Pontzer, C. H. et al. (1991)Cancer Res. 51:5304; Oldham, 1985, supra).

Hepatitis C virus (HCV) is a nonhost integrated RNA virus with a veryhigh rate of replication and is therefore associated with a large degreeof genetic diversity. At least six genotypes and more than thirtysubtypes of HCV RNA have been identified. HCV genotype has been shown tobe a predictor of response to IFN-alpha therapy. Patients infected withHCV genotypes 2 and 3 have been found to generally respond well tointerferon therapy. Patients infected with genotypes 4, 5 and 6 tend torespond less well. Patients infected with HCV genotype 1 tend to respondvery poorly to interferon therapy, with about 50% of Genotype 1 patientsclassified as “nonresponders” towards IFN-alpha therapy. Genotype 1 iscurrently the most prevalent form of Hepatitis C, infectingapproximately 70% of patients in the US and 50% of patients in Europe.Clearly, there is a pressing need for more effective therapies for HCVinfection, particularly of the Genotype 1 variety.

There is genetic and biochemical evidence that Genotype 1 HCV (and othersubtypes) actively attenuate the IFN-alpha signaling pathway byinhibiting key IFN responsive proteins such as the dsRNA-activatedserine/threonine protein kinase PKR (Katze M. G., et al. (2002) Nat.Rev. Immunol. 2(9):675-687). As a likely consequence of this geneticdiversity and active inhibition of the antiviral response, HCV(particularly Genotype 1) has the ability to escape the host's immunesurveillance, leading to a high rate of chronic infection. The extensivegenetic heterogeneity of HCV has important diagnostic and clinicalimplications, potentially accounting for variations in clinical course,difficulties in vaccine development, and lack of response to therapy.

The present invention addresses the need for interferon-alpha moleculeswhich exhibit enhanced antiviral efficacy and/or enhancedimmunomodulatory efficacy compared to interferon-alphas currently inclinical use. The invention provides novel interferon-alpha polypeptidesand polypeptide conjugates, nucleic acids encoding the polypeptides, andmethods of using such molecules. Such molecules would be of beneficialuse in a variety of applications, including, e.g., therapeutic andprophylactic treatments, particularly for viral infections and diseasesand conditions associated with viral infections. The present inventionfulfills these and other needs.

SUMMARY OF THE INVENTION

The present invention provides novel polypeptides, including variantsand fusion polypeptides. The invention also provides conjugatescomprising a polypeptide of the invention covalently linked to one ormore non-polypeptide moieties. The invention also provides nucleic acidsencoding any of the polypeptides of the invention, and vectors and hostcells comprising such nucleic acids. In addition, the invention providesmethods of making and using such polypeptides, conjugates, and nucleicacids, and other features apparent upon further review.

In one aspect, the invention provides an isolated or recombinantpolypeptide, the polypeptide comprising a sequence identified as one ofSEQ ID NOs:1-15 and SEQ ID NOs:44-104 (such as one of SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:44, SEQ IDNO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ IDNO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ IDNO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ IDNO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ IDNO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ IDNO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ IDNO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ IDNO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ IDNO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ IDNO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ IDNO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ IDNO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, and SEQ ID NO:104).

The invention also provides isolated or recombinant polypeptides whicheach comprise a sequence which differs in 0-16 amino acid positions(such as in 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16amino acid positions), e.g. in 0-14 amino acid positions, in 0-12 aminoacid positions, in 0-10 amino acid positions, in 0-8 amino acidpositions, in 0-6 amino acid positions, in 0-5 amino acid positions, in0-4 amino acid positions, in 0-3 amino acid positions, in 0-2 amino acidpositions, or in 0-1 amino acid position, from one of SEQ ID NOs:1-15and SEQ ID NOs:44-104, such as, for example, one of SEQ ID NOs:1-15, 47,or 53 (for example, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12,SEQ ID NO:47, or SEQ ID NO:53). In some instances, the polypeptideexhibits an interferon-alpha activity (such as, e.g., antiviralactivity, T_(H)1 differentiation activity, and/or antiproliferativeactivity). In some instances, the polypeptide sequence comprises asubstitution at one or more of positions 47, 51, 52, 53, 54, 55, 56, 57,58, 60, 61, 64, 69, 71, 72, 75, 76, 77, 78, 79, 80, 83, 84, 85, 86, 87,90, 93, 133, 140, 154, 160, 161, and 162, relative to one of SEQ ID NOs:1-15, 47, or 53, such as, for example, one of SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53. In someinstances, the polypeptide sequence comprises one or more of: His or Glnat position 47; Val, Ala or Thr at position 51; Gln, Pro or Glu atposition 52; Ala or Thr at position 53; Phe, Ser, or Pro at position 55;Leu, Val or Ala at position 56; Phe or Leu at position 57; Tyr or His atposition 58; Met, Leu or Val at position 60; Met or Ile at position 61;Thr or Ile at position 64; Ser or Thr at position 69; Lys or Glu atposition 71; Asn or Asp at position 72; Ala or Val at position 75; Alaor Thr at position 76; Trp or Leu at position 77; Asp or Glu at position78; Glu or Gln at position 79; Thr, Asp, Ser, or Arg at position 80; Gluor Asp at position 83; Lys or Glu at position 84; Phe or Leu at position85; Tyr, Cys or Ser at position 86; Ile or Thr at position 87; Phe, Tyr,Asp or Asn at position 90; Met or Leu at position 93; Lys or Glu atposition 133; Ser or Ala at position 140; Phe or Leu at position 154;Lys or Glu at position 160; Arg or Ser at position 161; and Arg or Serat position 162; the position numbering relative to that of SEQ ID NO:1.In some instances, the polypeptide sequence comprises one or more ofHis47, Val51, Phe55, Leu56, Tyr58, Lys133, and Ser140, the positionnumbering relative to that of SEQ ID NO:1. Some such polypeptidesinclude SEQ ID NOs:1-15 and SEQ ID NOs:44-104. The invention alsoprovides fusion proteins and conjugates comprising any of thesepolypeptides, nucleic acids encoding such polypeptides, and methods ofmaking such polypeptides.

Some polypeptides of the invention comprise one or more substitution,including but not limited to a substitution selected from: D2C, L3C,P4C, Q5C, T6C, H7C, S8C, L9C, G10C, R12C, R13C, M16C, A19C, Q20C, R22C,R23C, 124C, S25C, L26C, F27C, S28C, L30C, K31C, R33C, H34C, D35C, R37C,Q40C, E41C, E42C, D44C, N46C, H47C, Q49C, K50C, V51C, Q52C, E59C, Q62C,Q63C, N66C, S69C, T70C, K71C, N72C, S74C, A75C, D78C, E79C, T80C, L81C,E83C, K84C, 187C, F90C, Q91C, N94C, D95C, E97C, A98C, V100C, M101C,Q102C, E103C, V104C, G105C, E107C, E108C, T109C, P110C, L111C, M112C,N113C, V114C, D115C, L118C, R121C, K122C, Q125C, R126C, T128C, L129C,T132C, K133C, K134C, K135C, Y136C, S137C, P138C, A146C, M149C, R150C,S153C, F154C, N157C, Q159C, K160C, R161C, L162C, R163C, R164C, K165C andE166C (or equivalent position relative to SEQ ID NO:1), and combinationsthereof.

Some polypeptides of the invention comprise one or more substitution,including but not limited to a substitution selected from: D2K, L3K,P4K, Q5K, T6K, H7K, S8K, L9K, G10K, R12K, R13K, M16K, A19K, Q20K, R22K,R23K, I24K, S25K, L26K, F27K, S28K, L30K, R33K, H34K, D35K, R37K, Q40K,E41K, E42K, D44K, N46K, H47K, Q49K, V51K, Q52K, E59K, Q62K, Q63K, N66K,S69K, T70K, N72K, S74K, A75K, D78K, E79K, T80K, L81K, E83K, 187K, F90K,Q91K, N94K, D95K, E97K, A98K, V100K, M101K, Q102K, E103K, V104K, G105K,E107K, E108K, T109K, P110K, L111K, M112K, N113K, V114K, D115K, L118K,R121K, Q125K, R126K, T128K, L129K, T132K, Y136K, S137K, P138K, A146K,M149K, R150K, S153K, F154K, N157K, Q159K, R161K, L162K, R163K, R164K,and E166K (or equivalent position relative to SEQ ID NO:1), andcombinations thereof.

Some polypeptides of the invention comprise one or more substitution ofan amino acid residue for a different amino acid residue, or one or moredeletion of an amino acid residue, which removes one or more lysines,e.g., K31, K50, K71, K84, K122, K133, K134, K135, K160, and/or K165(relative to SEQ ID NO:1) from any polypeptide of the invention such as,for example, any one of SEQ ID NOs:1-15 and SEQ ID NOs:44-104 (such as,e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47or SEQ ID NO:53). The one or more lysine residue(s) to be removed may besubstituted with any other amino acid, may be substituted with an Arg(R) or Gln (Q), or may be deleted.

Some polypeptides of the invention comprise one or more substitution ofan amino acid residue for a different amino acid residue, or one or moredeletion of an amino acid residue, which removes one or more histidines,e.g., H7, H11, H34, and/or H47 (relative to SEQ ID NO:1) from anypolypeptide of the invention such as, for example, one of SEQ IDNOs:1-15 and SEQ ID NOs:44-104 (such as, e.g., SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53). The one ormore histidine residue(s) to be removed may be substituted with anyother amino acid, may be substituted with an Arg (R) or Gln (Q), or maybe deleted.

Some polypeptides of the invention comprise one or more substitution,including but not limited to substitutions selected from: D2N+P4S/T,L3N+Q5S/T, P4Q, P4Q+T6S, Q5N+H7S/T, T6N, T6N+S8T, H7N+L9S/T, S8N+G10S/T,L9N+H11S/T, G10N+R12S/T, R12N, R12N+T14S, R13N+M15S/T, M16N+L18S/T,A19N+M21S/T, Q20N+R22S/T, R22N+124S/T, R23N, R23N+S25T, I24N+L26S/T,S25N+F27S/T, L26N, L26N+S28T, S28N+L30S/T, L30N+D32S/T, K31N+R33S/T,R33N+D35S/T, H34N+F36S/T, D35N+R37S/T, R37N+P39S/T, Q40N+E42S/T,E41N+F43S/T, E42N+D44S/T, D44N+N46S/T, F48S/T, H47N+Q49S/T, Q49N+V51S/T,K50N+Q52S/T, V51N+A53S/T, Q52N+154S/T, E59N+M61S/T, Q62N, Q62N+T64S,Q63N+F65S/T, F68S/T, S69N+K71S/T, T70N+N72S/T, K71N, K71N+S73T, S74T,S74N+A76S/T, A75N+W77S/T, D78N, D78N+T80S, E79N+L81S/T, T80N+L82S/T,L81N+E83S/T, E83N+F85S/T, K84N+Y86S/T, 187N+L89S/T, F90N+Q92S/T,Q91N+M93S/T, L96S/T, D95N+E97S/T, E97N+C99S/T, A98N+V100S/T,V100N+Q102S/T, M101N+E103S/T, Q102N+V1014S/T, E103N+G105S/T,V104N+V106S/T, G105N+E107S/T, E107, E107N+T109S, E108N+P110S/T,L111N+N113S/T, M112N+V114S/T, N113N+D115S/T, V114N, V114N+S116T,D115N+1117S/T, L118N+V120S/T, R121N+Y123S/T, K122N+F124S/T,Q125N+1127S/T, R126N, R126N+T128S, T128N+Y130S/T, L129N+L131S/T,T132N+K134S/T, K133N+K135S/T, K134N+Y136S/T, K135N, K135N+S137T,Y136N+P138S/T, P138N, P138N+S140T, A146N+1148S/T, M149N, M149N+S151T,R150N+F152S/T, S153N, S153N+S155T, F154N+F156S/T, Q159S/T,K160N+L162S/T, R161N+R163S/T, L162N+R164S/T, R163N+K165S/T andR164N+E166S/T (or equivalent positions relative to SEQ ID NO:1), andcombinations thereof.

The invention also provides fusion proteins and conjugates comprisingany of the above polypeptides, nucleic acids encoding such polypeptides,and methods of making and using such polypeptides.

In another aspect, the invention provides isolated or recombinantpolypeptides which each comprise a sequence having at least about 90%amino acid sequence identity to one of SEQ ID NOs:1-15 and SEQ IDNOs:44-104, such as, for example, one of SEQ ID NOs:1-15, 47, and 53(such as, for example, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ IDNO:12, SEQ ID NO:47, or SEQ ID NO:53). Some such polypeptides exhibit aninterferon-alpha activity (such as, e.g., antiviral activity, T_(H)1differentiation activity, and/or antiproliferative activity). In someinstances, the sequence has at least about 91%, 92%, 93%, 94%, 95%, 96%,97%, 98% or 99% identity to one of SEQ ID NOs:1-15 and SEQ ID NOs 44-104(such as, e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQID NO:47, or SEQ ID NO:53). In some instances, the polypeptide sequencecomprises a substitution at one or more of positions 47, 51, 52, 53, 54,55, 56, 57, 58, 60, 61, 64, 69, 71, 72, 75, 76, 77, 78, 79, 80, 83, 84,85, 86, 87, 90, 93, 133, 140, 154, 160, 161, and 162, relative to, e.g.,one of SEQ ID NOs:1-15. In some instances, the polypeptide sequencecomprises one or more of: His or Gln at position 47; Val, Ala or Thr atposition 51; Gln, Pro or Glu at position 52; Ala or Thr at position 53;Phe, Ser, or Pro at position 55; Leu, Val or Ala at position 56; Phe orLeu at position 57; Tyr or His at position 58; Met, Leu or Val atposition 60; Met or Ile at position 61; Thr or Ile at position 64; Seror Thr at position 69; Lys or Glu at position 71; Asn or Asp at position72; Ala or Val at position 75; Ala or Thr at position 76; Trp or Leu atposition 77; Asp or Glu at position 78; Glu or Gln at position 79; Thr,Asp, Ser, or Arg at position 80; Glu or Asp at position 83; Lys or Gluat position 84; Phe or Leu at position 85; Tyr, Cys or Ser at position86; Ile or Thr at position 87; Phe, Tyr, Asp or Asn at position 90; Metor Leu at position 93; Lys or Glu at position 133; Ser or Ala atposition 140; Phe or Leu at position 154; Lys or Glu at position 160;Arg or Ser at position 161; and Arg or Ser at position 162 (the positionnumbering relative to that of SEQ ID NO:1). In some instances, thepolypeptide sequence comprises one or more of His47, Val51, Phe55,Leu56, Tyr58, Lys133, and Ser140, the position numbering relative tothat of SEQ ID NO:1. Some such polypeptides comprise a sequence selectedfrom SEQ ID NOs:1-15 and SEQ ID NOs:44-104. The invention also providesfusion proteins and conjugates comprising any of the above polypeptides,nucleic acids encoding such polypeptides, and methods of making suchpolypeptides.

In another aspect, the invention provides isolated or recombinantpolypeptides which are variants of a parent polypeptide, each variantcomprising a variant sequence which differs from the parent polypeptidesequence in least one amino acid position relative to the parentpolypeptide sequence, wherein the parent polypeptide sequence is one ofSEQ ID NOs:1-15 and SEQ ID NOs:44-104, such as, for example, one of SEQID NOs:1-15, 47, and 53, e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8,SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53. In some instances, thevariant exhibits an interferon-alpha activity (such as, antiviralactivity, T_(H)1 differentiation activity, and/or antiproliferativeactivity). In some instances, the variant sequence differs from theparent polypeptide sequence in 1-16 amino acid positions (such as in 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acidpositions), e.g. in 1-14 amino acid positions, in 1-12 amino acidpositions, in 1-10 amino acid positions, in 1-8 amino acid positions, in1-6 amino acid positions, in 1-5 amino acid positions, in 1-4 amino acidpositions, in 1-3 amino acid positions, or in 1-2 amino acid positions.In some instances, the variant sequence comprises a substitution at oneor more of positions 47, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 64, 69,71, 72, 75, 76, 77, 78, 79, 80, 83, 84, 85, 86, 87, 90, 93, 133, 140,154, 160, 161, and 162, relative to one of SEQ ID NOs:1-15. In someinstances, the variant sequence comprises one or more of: His or Gln atposition 47; Val, Ala or Thr at position 51; Gln, Pro or Glu at position52; Ala or Thr at position 53; Phe, Ser, or Pro at position 55; Leu, Valor Ala at position 56; Phe or Leu at position 57; Tyr or His at position58; Met, Leu or Val at position 60; Met or Ile at position 61; Thr orIle at position 64; Ser or Thr at position 69; Lys or Glu at position71; Asn or Asp at position 72; Ala or Val at position 75; Ala or Thr atposition 76; Trp or Leu at position 77; Asp or Glu at position 78; Gluor Gln at position 79; Thr, Asp, Ser, or Arg at position 80; Glu or Aspat position 83; Lys or Glu at position 84; Phe or Leu at position 85;Tyr, Cys or Ser at position 86; Ile or Thr at position 87; Phe, Tyr, Aspor Asn at position 90; Met or Leu at position 93; Lys or Glu at position133; Ser or Ala at position 140; Phe or Leu at position 154; Lys or Gluat position 160; Arg or Ser at position 161; and Arg or Ser at position162; the position numbering relative to that of SEQ ID NO:1. In someinstances, the variant sequence comprises one or more of His47, Val51,Phe55, Leu56, Tyr58, Lys133, and Ser140, the position numbering relativeto that of SEQ ID NO:1. Some such variants comprise a sequence selectedfrom SEQ ID NOs:1-15 and SEQ ID NOs:44-104. The invention also providesfusion proteins and conjugates comprising any of these variants, nucleicacids encoding any of these variants, and methods of making suchvariants.

In another aspect, the invention provides isolated or recombinantpolypeptides which are variants of a parent interferon-alphapolypeptide, each variant comprising a variant sequence which differsfrom the parent interferon-alpha polypeptide sequence in least one aminoacid position, wherein the variant sequence comprises one or more ofHis47, Val51, Phe55, Leu56, Tyr58, Lys133, and Ser140, the positionnumbering relative to that of SEQ ID NO:1. In some instances the parentinterferon-alpha polypeptide sequence is a sequence of anaturally-occurring human interferon-alpha, such as one of SEQ IDNO:31-SEQ ID NO:42 or SEQ ID NO:32+R23K, or a non-naturally occurring(i.e., synthetic) interferon-alpha, such as SEQ ID NO:43. In someinstances, the variant sequence differs from the parent interferon-alphapolypeptide sequence in 1-16 amino acid positions (such as in 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid positions),e.g. in 1-14 amino acid positions, in 1-12 amino acid positions, in 1-10amino acid positions, in 1-8 amino acid positions, in 1-6 amino acidpositions, in 1-5 amino acid positions, in 1-4 amino acid positions, in1-3 amino acid positions, or in 1-2 amino acid positions. In someinstances, the variant exhibits an interferon-alpha activity (such as,e.g., antiviral activity, T_(H)1 differentiation activity, and/orantiproliferative activity). The invention also provides fusion proteinsand conjugates comprising any of these variants, nucleic acids encodingany of these variants, and methods of making such variants.

The invention also provides conjugates comprising a polypeptide of theinvention, such as any of the polypeptides of the invention (includingvariants) described above, and at least one non-polypeptide moietyattached to an attachment group of the polypeptide, wherein theconjugate exhibits an interferon-alpha activity. In some instances, thenon-polypeptide moiety is a polymer (such as, e.g., PEG or mPEG), or asugar moiety. The at least one non-polypeptide moiety may be attached toa cysteine, to a lysine, to the N-terminal amino group of thepolypeptide, to an in vivo glycosylation site of the polypeptide. Theinvention also provides methods of making and using such conjugates.

The invention also provides isolated or recombinant nucleic acidsencoding any of the polypeptides (including variants) of the invention.The invention also provides vectors and host cells comprising suchnucleic acids, and methods of making polypeptides of the invention,comprising culturing host cells comprising such nucleic acids.

In another aspect, the invention provides a method of inhibiting viralreplication in virus-infected cells, the method comprising contactingthe virus-infected cells with a polypeptide or a conjugate of theinvention. The invention also provides a method of reducing the numberof copies of a virus in virus-infected cells, comprising contacting thevirus-infected cells with a polypeptide or a conjugate of the invention.

In another aspect, the invention provides a method for reducing thelevel of a virus in the serum of a patient infected with the virus,comprising administering to the patient the polypeptide or a conjugateof the invention in an amount effective to reduce the level of the virusin the serum compared to the level present prior to the start oftreatment.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show biphasic timecourses for viral clearance fromHCV-infected cells following IFN-alpha treatment (A. Nonresponderkinetics; B. Responder kinetics).

FIG. 2 shows an alignment of the sequence of a polypeptide of theinvention (SEQ ID NO: 1) with the following human interferon-alphapolypeptide sequences: huIFN-alpha 1a (SEQ ID NO:31), huIFN-alpha 2b(SEQ ID NO:32), huIFN-alpha 4b (SEQ ID NO:33), huIFN-alpha 5 (SEQ IDNO:34), huIFN-alpha 6 (SEQ ID NO:35), huIFN-alpha 7 (SEQ ID NO:36),huIFN-alpha 8b (SEQ ID NO:37), huIFN-alpha 10a (SEQ ID NO:38),huIFN-alpha 14a (SEQ ID NO:39), huIFN-alpha 16 (SEQ ID NO:40),huIFN-alpha 17b (SEQ ID NO:41) and huIFN-alpha 21b (SEQ ID NO:42). Thenaming conventions for the huIFN-alpha sequences are according to AllenG. and Diaz M. O. (1996) J. Interferon and Cytokine Res. 16:181-184. Thearrows indicate residues His47, Val51, Phe55, Leu56, Tyr58, Lys133, andSer140 of SEQ ID NO:1, which are not present in any of SEQ ID NOs:31-SEQID NO:42. Amino acid residue positions in SEQ ID NOs:31-42 which areidentical to SEQ ID NO:1 are indicated with a period (.), and gaps inthe sequence are indicated with a dash (-).

FIG. 3 shows an alignment of the sequence of a polypeptide of theinvention (SEQ ID NO:3) with huIFN-alpha 14a (SEQ ID NO:39) (LeIF H;Goeddel et al. (1981) Nature 290:20-26) using the following parameters:BLOSUM62 matrix, gap open penalty 11, gap extension penalty 1. Aminoacid positions in SEQ ID NO:39 which are identical to SEQ ID NO:3 areindicated with a period (.).

FIG. 4 shows an alignment of the sequence of a polypeptide of theinvention (SEQ ID NO:8) with human interferon-alpha polypeptidesequences SEQ ID NO:31-SEQ ID NO:42. Amino acid residue positions in SEQID NOs:31-42 which are identical to SEQ ID NO:8 are indicated with aperiod (.), and gaps in the sequence are indicated with a dash (-).

FIG. 5 shows an alignment of the sequence of a polypeptide of theinvention (SEQ ID NO: 12) with huIFN-alpha 14a (SEQ ID NO:39) (LeIF H;Goeddel et al. (1981) Nature 290:20-26) using the following parameters:BLOSUM62 matrix, gap open penalty 11, gap extension penalty 1. Aminoacid positions in SEQ ID NO:39 which are identical to SEQ ID NO:12 areindicated with a period (.).

FIG. 6 shows the BLOSUM62 substitution matrix.

FIGS. 7A, 7B and 7C show examples of calculations of alignment scoresused to determine optimal sequence alignments, using the followingparameters: BLOSUM62 matrix, gap open penalty=11, and gap extensionpenalty=1.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise defined herein or in the remainder of thespecification, all technical and scientific terms used herein have thesame meaning as commonly understood by those of ordinary skill in theart to which the invention belongs.

A “polypeptide sequence” (e.g., a protein, polypeptide, peptide, etc.)is a polymer of amino acids comprising naturally occurring amino acidsor artificial amino acid analogues, or a character string representingan amino acid polymer, depending on context. Given the degeneracy of thegenetic code, one or more nucleic acids, or the complementary nucleicacids thereof, that encode a specific polypeptide sequence can bedetermined from the polypeptide sequence.

A “polynucleotide sequence” (e.g., a nucleic acid, polynucleotide,oligonucleotide, etc.) is a polymer of nucleotides comprisingnucleotides A, C, T, U, G, or other naturally occurring nucleotides orartificial nucleotide analogues, or a character string representing anucleic acid, depending on context. Either the given nucleic acid or thecomplementary nucleic acid can be determined from any specifiedpolynucleotide sequence.

Numbering of a given amino acid polymer or nucleic acid polymer“corresponds to” or is “relative to” the numbering of a selected aminoacid polymer or nucleic acid polymer when the position of any givenpolymer component (e.g., amino acid, nucleotide, also referred togenerically as a “residue”) is designated by reference to the same or anequivalent position in the selected amino acid or nucleic acid polymer,rather than by the actual numerical position of the component in thegiven polymer. Thus, for example, the numbering of a given amino acidposition in a given polypeptide sequence corresponds to the same orequivalent amino acid position in a selected polypeptide sequence usedas a reference sequence.

An “equivalent position” (for example, an “equivalent amino acidposition” or “equivalent residue position”) is defined herein as aposition (such as, an amino acid position or a residue position) of atest polypeptide sequence which aligns with a corresponding position ofa reference polypeptide sequence, using an alignment algorithm asdescribed herein. The equivalent amino acid position of the testpolypeptide sequence need not have the same numerical position number asthe corresponding position of the test polypeptide. As an example, FIG.2 shows the sequence of a polypeptide of the invention (SEQ ID NO:1)aligned with various known human interferon-alpha polypeptide sequences.In this example, amino acid position number 47 of SEQ ID NO:1 isconsidered to be an equivalent amino acid position to (i.e. is“equivalent to”) that of amino acid position number 46 of SEQ ID NO:32(huIFN-alpha 2b), since amino acid number 47 of SEQ ID NO:1 aligns withamino acid number 46 of SEQ ID NO:32. In other words, amino acidposition 47 of SEQ ID NO:1 Corresponds to amino acid position 46 of SEQID NO:32. Likewise, residue H47 in SEQ ID NO:1 is understood tocorrespond to residue Q47 in SEQ ID NO:5, so that for example thesubstitution H47C relative to SEQ ID NO:1 is understood to correspond tothe substitution Q47C in, e.g., SEQ ID NO:5 (and so on).

Two polypeptide sequences are “optimally aligned” when they are alignedusing defined parameters, i.e., a defined amino acid substitutionmatrix, gap existence penalty (also termed gap open penalty), and gapextension penalty, so as to arrive at the highest similarity scorepossible for that pair of sequences. The BLOSUM62 matrix (Henikoff andHenikoff (1992) Proc. Natl. Acad. Sci. USA 89(22):10915-10919) is oftenused as a default scoring substitution matrix in polypeptide sequencealignment algorithms (such as BLASTP). The gap existence penalty isimposed for the introduction of a single amino acid gap in one of thealigned sequences, and the gap extension penalty is imposed for eachresidue position in the gap. Unless otherwise stated, alignmentparameters employed herein are: BLOSUM62 scoring matrix, gap existencepenalty=11, and gap extension penalty=1. The alignment score is definedby the amino acid positions of each sequence at which the alignmentbegins and ends (e.g. the alignment window), and optionally by theinsertion of a gap or multiple gaps into one or both sequences, so as toarrive at the highest possible similarity score, as described in moredetail below in the section entitled “Percent Sequence Identity”.

The terminology used for identifying amino acid positions and amino acidsubstitutions is illustrated as follows: H47 indicates position number47 occupied by a histidine (H is) residue in a reference amino acidsequence, e.g. SEQ ID NO:1. H47Q indicates that the histidine residue ofposition 47 has been substituted with a glutamine (Gln) residue.Alternative substitutions are indicated with a “/”, e.g., H47S/T meansan amino acid sequence in which the histidine residue in position 47 issubstituted with a serine or a threonine residue. Multiple substitutionsmay be indicated with a “+”, e.g. H47Q+V51 S/T means an amino acidsequence which comprises a substitution of the histidine residue atposition 47 with an glutamine residue and a substitution of the valineresidue at position 51 with a serine or a threonine residue. Deletionsare indicated by an asterix. For example, H47* indicates that thehistidine residue in position 47 has been deleted. Deletions of two ormore continuous amino acids may be indicated as follows, e.g.,R161*-E166* indicates the deletion of residues R161-E166 inclusive (thatis, residues 161, 162, 163, 164, 164, and 166 are deleted). Insertionsare indicated the following way: Insertion of an additional serineresidue after the histidine residue located at position 47 is indicatedas H47HS. Combined substitutions and insertions are indicated in thefollowing way: Substitution of the histidine residue at position 47 witha serine residue and insertion of an alanine residue after the position47 amino acid residue is indicated as H47SA.

Unless otherwise indicated, the position numbering of amino acidresidues recited herein is relative to the amino acid sequence SEQ IDNO:1. It is to be understood that while the examples and modificationsto the parent polypeptide are generally provided herein relative to thesequence SEQ ID NO:1 (or relative to another specified sequence), theexamples pertain to other polypeptides of the invention, and themodifications described herein may be made in equivalent amino acidpositions of any of the other polypeptides described herein. Thus, as anexample, the substitution H47C relative to SEQ ID NO:1 is understood tocorrespond to the substitution Q47C in SEQ ID NO:5, and so on.

The term “exhibiting (or exhibits, or having, or has) aninterferon-alpha activity” is intended to indicate that the polypeptideor conjugate of the invention has at least one activity exhibited by areference interferon-alpha polypeptide (such as, for example, a humaninterferon-alpha polypeptide, e.g., huIFN-alpha 2b identified herein asSEQ ID NO:32, huIFN-alpha 2a identified herein as SEQ ID NO:32+R23K,hIFN-alpha 8b identified herein as SEQ ID NO:37, or any other humaninterferon alpha polypeptide known in the art, such as, for example,those shown in FIGS. 2 and 4 herein and/or listed in Allen G. and DiazM. O. (1996), supra). Such activity includes the ability to signalthrough an interferon-alpha receptor, as evidenced by, for example, oneor more of: inhibition of viral replication in virus-infected cells(“antiviral activity”); enhancement of differentiation of naïve T-cellsto a T_(H)1 phenotype and/or suppression of differentiation of naïveT-cells to a T_(H)2 phenotype (“T_(H)1 differentiation activity”); orinhibition of cell proliferation (“antiproliferative activity”). The oneor more interferon-alpha activity is assayed using assays known in theart and/or described in the Examples.

A polypeptide or a conjugate exhibiting an interferon-alpha activity isconsidered to have such activity when it displays a measurable activity,e.g., a measurable antiviral activity, antiproliferative activity, orT_(H)1 differentiation activity (e.g., as determined by assays known inthe art and/or described in the Examples). One of skill in the artrecognizes that what constitutes a measurable activity depends in parton the nature of the assay being undertaken, but as a general guidelinea measurable activity is one in which the assay signal generated in thepresence of the test compound (e.g., a polypeptide of the invention) isquantifiably different than the assay signal generated in the absence ofthe test compound. It is to be understood that the polypeptide orconjugate of the invention need not exhibit all of the known activitiesof a particular reference interferon-alpha, or exhibit such activitiesto the same extent as the reference interferon-alpha. In some instancesthe activity exhibited by a polypeptide or conjugate of the invention(as evidenced, e.g., by an EC₅₀, specific activity, or other valuerelated to activity) may be about equal to, be less than, or be greaterthan that of the particular activity exhibited by the referenceinterferon-alpha.

A “variant” is a polypeptide comprising a sequence which differs in oneor more amino acid position(s) from that of a parent polypeptidesequence. For example, a variant may comprise a sequence which differsfrom the parent polypeptides sequence in up to 10% of the total numberof residues of the parent polypeptide sequence, such as in up to 8% ofthe residues, e.g., in up to 5%, 4%, 3% 2% or 1% of the total number ofresidue of the parent polypeptide sequence. For example, a variant ofSEQ ID NO:1 may comprise a sequence which differs from SEQ ID NO:1 in1-16 amino acid positions (such as in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, or 16 amino acid positions), e.g. in 1-15 amino acidpositions, in 1-14 amino acid positions, in 1-13 amino acid positions,in 1-12 amino acid positions, in 1-11 amino acid positions, in 1-10amino acid positions, in 1-9 amino acid positions, in 1-8 amino acidpositions, in 1-7 amino acid positions, in 1-6 amino acid positions, in1-5 amino acid positions, in 1-4 amino acid positions, in 1-3 amino acidpositions, or in 1-2 amino acid positions.

The term “parent polypeptide” or “parent interferon-alpha” is intendedto indicate the polypeptide sequence to be modified in accordance withthe present invention. The parent polypeptide sequence may be that of anaturally occurring IFN-alpha (such as a mammalian IFN-alpha, e.g., aprimate IFN-alpha, such as a human IFN-alpha, such as a huIFN-alphapolypeptide identified herein as SEQ ID NOs:31-42, SEQ ID NO:32+R23K, orother huIFN-alpha sequence described herein and/or in Allen G. and DiazM. O. (1996), supra). The parent polypeptide sequence may be that of anon-naturally occurring (i.e., “synthetic”) interferon-alpha, such asIFN-alpha Con1 (SEQ ID NO:43). In some instances, the parent polypeptideto be modified may itself be a polypeptide of the invention, such as,e.g. any one of SEQ ID NOs:1-15 and SEQ ID NOs:44-104.

“Naturally occurring” as applied to an object refers to the fact thatthe object can be found in nature as distinct from being artificiallyproduced by man. For example, a polypeptide or polynucleotide sequencethat is present in an organism (including viruses, bacteria, protozoa,insects, plants or mammalian tissue) that can be isolated from a sourcein nature and which has not been intentionally modified by man in thelaboratory is naturally occurring. “Non-naturally occurring” (alsotermed “synthetic” or “artificial”) as applied to an object means thatthe object is not naturally-occurring—i.e., the object cannot be foundin nature as distinct from being artificially produced by man.

A “fragment” or “subsequence” is any portion of an entire sequence, upto but not including the entire sequence. Thus, a fragment orsubsequence refers to a sequence of amino acids or nucleic acids thatcomprises a part of a longer sequence of amino acids (e.g., polypeptide)or nucleic acids (e.g., polynucleotide).

One type of fragment contemplated by the present invention is a fragmentin which amino acid residues are removed from the N-terminus or theC-terminus of the parent polypeptide (or both); such a polypeptide isconsidered to be “N-terminally truncated” or “C-terminally truncated”,respectively. It is known that deletion of at least the first four aminoacids from the N-terminus does not significantly affect interferon-alphaactivity (Lydon, N. B. et al. (1985) Biochemistry 24: 4131-41).Furthermore, variants retaining interferon-alpha activity have beendescribed wherein between 7 and 11 amino acids have been deleted fromthe C-terminus (Cheetham B. F. et al. (1991) Antiviral Res. 15(1):27-39;Chang N. T. et al. (1983) Arch. Biochem Biophys. 221(2): 585-589; FrankeA. E. et al. (1982) DNA 1(3):223-230).

A “receptor” e.g., an “interferon-alpha receptor” (also known as a “TypeI interferon receptor”) is a receptor which is activated in cells by aninterferon-alpha, e.g., binds an interferon-alpha and initiatesintracellular signaling, such as a type I interferon receptor comprisingreceptor subunits IFNAR-2 and IFNAR-1 (Domanski et al. (1998) J. Biol.Chem. 273(6):3144-3147; Mogensen et al., (1999) Journal of Interferonand Cytokine Research, 19:1069-1098). In the context of this invention,receptor is also meant to include truncated forms of a full-lengthreceptor molecule, such as for example a receptor molecule which lacks amembrane-binding portion, such as a soluble form of a receptor molecule(also known as a “soluble receptor”) which comprises an extracelluarbinding domain, which binds an interferon-alpha, but may not necessarilybind to a membrane and/or initiate intracellular signaling.

A “specific binding affinity” between two molecules, e.g., a ligand anda receptor, means a preferential binding of one molecule for another ina mixture of molecules. The binding of the molecules is typicallyconsidered specific if the binding affinity is about 1×10⁴ M⁻¹ to about1×10⁹ M⁻¹ or greater (i.e., K_(D) of about 10⁻⁴ to 10⁻⁹ M or less).Binding affinity of a ligand and a receptor may be measured by standardtechniques known to those of skill in the art. Non-limiting examples ofwell-known techniques for measuring binding affinities include Biacore®technology (Biacore AB, Sweden), isothermal titration microcalorimetry(MicroCal LLC, Northampton, Mass. USA), ELISA, and FACS. For example,FACS or other sorting methods may be used to select for populations ofmolecules (such as for example, cell surface-displayed ligands) whichspecifically bind to the associated binding pair member (such as areceptor, e.g., a soluble receptor). Ligand-receptor complexes may bedetected and sorted e.g., by fluorescence (e.g., by reacting the complexwith a fluorescent antibody that recognizes the complex). Molecules ofinterest which bind an associated binding pair member (e.g., receptor)are pooled and re-sorted in the presence of lower concentrations ofreceptor. By performing multiple rounds sorting in the presence ofdecreasing concentrations of receptor (an exemplary concentration rangebeing on the order of 10⁻⁶ M down to 10⁻⁹ M, i.e., 1 micromolar (μM)down to 1 nanomolar (nM), or less, depending on the nature of theligand-receptor interaction), populations of the molecule of interestexhibiting specific binding affinity for the receptor may be isolated.

A polypeptide, nucleic acid, or other component is “isolated” when it ispartially or completely separated from components with which it isnormally associated (other peptides, polypeptides, proteins (includingcomplexes, e.g., polymerases and ribosomes which may accompany a nativesequence), nucleic acids, cells, synthetic reagents, cellularcontaminants, cellular components, etc.), e.g., such as from othercomponents with which it is normally associated in the cell from whichit was originally derived. A polypeptide, nucleic acid, or othercomponent is isolated when it is partially or completely recovered orseparated from other components of its natural environment such that itis the predominant species present in a composition, mixture, orcollection of components (i.e., on a molar basis it is more abundantthan any other individual species in the composition). In someinstances, the preparation consists of more than about 60%, 70% or 75%,typically more than about 80%, or preferably more than about 90% of theisolated species.

A “substantially pure” or “isolated” nucleic acid (e.g., RNA or DNA),polypeptide, protein, or composition also means where the object species(e.g., nucleic acid or polypeptide) comprises at least about 50, 60, or70 percent by weight (on a molar basis) of all macromolecular speciespresent. A substantially pure or isolated composition can also compriseat least about 80, 90, or 95 percent by weight of all macromolecularspecies present in the composition. An isolated object species can alsobe purified to essential homogeneity (contaminant species cannot bedetected in the composition by conventional detection methods) whereinthe composition consists essentially of derivatives of a singlemacromolecular species. The term “purified” generally denotes that anucleic acid, polypeptide, or protein gives rise to essentially one bandin an electrophoretic gel. It typically means that the nucleic acid,polypeptide, or protein is at least about 50% pure, 60% pure, 70% pure,75% pure, more preferably at least about 85% pure, and most preferablyat least about 99% pure.

The term “isolated nucleic acid” may refer to a nucleic acid (e.g., DNAor RNA) that is not immediately contiguous with both of the codingsequences with which it is immediately contiguous (i.e., one at the 5′and one at the 3′ end) in the naturally occurring genome of the organismfrom which the nucleic acid of the invention is derived. Thus, this termincludes, e.g., a cDNA or a genomic DNA fragment produced by polymerasechain reaction (PCR) or restriction endonuclease treatment, whether suchcDNA or genomic DNA fragment is incorporated into a vector, integratedinto the genome of the same or a different species than the organism,including, e.g., a virus, from which it was originally derived, linkedto an additional coding sequence to form a hybrid gene encoding achimeric polypeptide, or independent of any other DNA sequences. The DNAmay be double-stranded or single-stranded, sense or antisense.

A “recombinant polynucleotide” or a “recombinant polypeptide” is anon-naturally occurring polynucleotide or polypeptide which may includenucleic acid or amino acid sequences, respectively, from more than onesource nucleic acid or polypeptide, which source nucleic acid orpolypeptide can be a naturally occurring nucleic acid or polypeptide, orcan itself have been subjected to mutagenesis or other type ofmodification. A nucleic acid or polypeptide may be deemed “recombinant”when it is synthetic or artificial or engineered, or derived from asynthetic or artificial or engineered polypeptide or nucleic acid. Arecombinant nucleic acid (e.g., DNA or RNA) can be made by thecombination (e.g., artificial combination) of at least two segments ofsequence that are not typically included together, not typicallyassociated with one another, or are otherwise typically separated fromone another. A recombinant nucleic acid can comprise a nucleic acidmolecule formed by the joining together or combination of nucleic acidsegments from different sources and/or artificially synthesized. A“recombinant polypeptide” often refers to a polypeptide that resultsfrom a cloned or recombinant nucleic acid. The source polynucleotides orpolypeptides from which the different nucleic acid or amino acidsequences are derived are sometimes homologous (i.e., have, or encode apolypeptide that encodes, the same or a similar structure and/orfunction), and are often from different isolates, serotypes, strains,species, of organism or from different disease states, for example.

The term “recombinant” when used with reference, e.g., to a cell,polynucleotide, vector, protein, or polypeptide typically indicates thatthe cell, polynucleotide, or vector has been modified by theintroduction of a heterologous (or foreign) nucleic acid or thealteration of a native nucleic acid, or that the protein or polypeptidehas been modified by the introduction of a heterologous amino acid, orthat the cell is derived from a cell so modified. Recombinant cellsexpress nucleic acid sequences that are not found in the native(non-recombinant) form of the cell or express native nucleic acidsequences that would otherwise be abnormally expressed, under-expressed,or not expressed at all. The term “recombinant” when used with referenceto a cell indicates that the cell replicates a heterologous nucleicacid, or expresses a polypeptide encoded by a heterologous nucleic acid.Recombinant cells can contain coding sequences that are not found withinthe native (non-recombinant) form of the cell. Recombinant cells canalso contain coding sequences found in the native form of the cellwherein the coding sequences are modified and re-introduced into thecell by artificial means. The term also encompasses cells that contain anucleic acid endogenous to the cell that has been modified withoutremoving the nucleic acid from the cell; such modifications includethose obtained by gene replacement, site-specific mutation,recombination, and related techniques.

The term “recombinantly produced” refers to an artificial combinationusually accomplished by either chemical synthesis means, recursivesequence recombination of nucleic acid segments or other diversitygeneration methods (such as, e.g., shuffling) of nucleotides, ormanipulation of isolated segments of nucleic acids, e.g., by geneticengineering techniques known to those of ordinary skill in the art.“Recombinantly expressed” typically refers to techniques for theproduction of a recombinant nucleic acid in vitro and transfer of therecombinant nucleic acid into cells in vivo, in vitro, or ex vivo whereit may be expressed or propagated.

A “recombinant expression cassette” or simply an “expression cassette”is a nucleic acid construct, generated recombinantly or synthetically,with nucleic acid elements that are capable of effecting expression of astructural gene in hosts compatible with such sequences. Expressioncassettes include at least promoters and optionally, transcriptiontermination signals. Typically, the recombinant expression cassetteincludes a nucleic acid to be transcribed (e.g., a nucleic acid encodinga desired polypeptide), and a promoter. Additional factors necessary orhelpful in effecting expression may also be used as described herein.For example, an expression cassette can also include nucleotidesequences that encode a signal sequence that directs secretion of anexpressed protein from the host cell. Transcription termination signals,enhancers, and other nucleic acid sequences that influence geneexpression, can also be included in an expression cassette.

An “immunogen” refers to a substance capable of provoking an immuneresponse, and includes, e.g., antigens, autoantigens that play a role ininduction of autoimmune diseases, and tumor-associated antigensexpressed on cancer cells. An immune response generally refers to thedevelopment of a cellular or antibody-mediated response to an agent,such as an antigen or fragment thereof or nucleic acid encoding suchagent. In some instances, such a response comprises a production of atleast one or a combination of CTLs, B cells, or various classes of Tcells that are directed specifically to antigen-presenting cellsexpressing the antigen of interest.

An “antigen” refers to a substance that is capable of eliciting theformation of antibodies in a host or generating a specific population oflymphocytes reactive with that substance. Antigens are typicallymacromolecules (e.g., proteins and polysaccharides) that are foreign tothe host.

An “adjuvant” refers to a substance that enhances an antigen'simmune-stimulating properties or the pharmacological effect(s) of adrug. An adjuvant may non-specifically enhance the immune response to anantigen. “Freund's Complete Adjuvant,” for example, is an emulsion ofoil and water containing an immunogen, an emulsifying agent andmycobacteria. Another example, “Freund's incomplete adjuvant,” is thesame, but without mycobacteria.

A vector is a component or composition for facilitating celltransduction or transfection by a selected nucleic acid, or expressionof the nucleic acid in the cell. Vectors include, e.g., plasmids,cosmids, viruses, YACs, bacteria, poly-lysine, etc. An “expressionvector” is a nucleic acid construct or sequence, generated recombinantlyor synthetically, with a series of specific nucleic acid elements thatpermit transcription of a particular nucleic acid in a host cell. Theexpression vector can be part of a plasmid, virus, or nucleic acidfragment. The expression vector typically includes a nucleic acid to betranscribed operably linked to a promoter. The nucleic acid to betranscribed is typically under the direction or control of the promoter.

“Substantially the entire length of a polynucleotide sequence” or“substantially the entire length of a polypeptide sequence” refers to atleast 50%, generally at least about 60%, 70%, or 75%, usually at leastabout 80%, or typically at least about 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% or more of a length of a polynucleotide sequenceor polypeptide sequence.

The term “immunoassay” includes an assay that uses an antibody orimmunogen to bind or specifically bind an antigen. The immunoassay istypically characterized by the use of specific binding properties of aparticular antibody to isolate, target, and/or quantify the antigen.

The term “subject” as used herein includes, but is not limited to, anorganism; a mammal, including, e.g., a human, non-human primate (e.g.,baboon, orangutan, monkey), mouse, pig, cow, goat, cat, rabbit, rat,guinea pig, hamster, horse, monkey, sheep, or other non-human mammal; anon-mammal, including, e.g., a non-mammalian vertebrate, such as a bird(e.g., a chicken or duck) or a fish, and a non-mammalian invertebrate.

The term “pharmaceutical composition” means a composition suitable forpharmaceutical use in a subject, including an animal or human. Apharmaceutical composition generally comprises an effective amount of anactive agent and a carrier, including, e.g., a pharmaceuticallyacceptable carrier.

The term “effective amount” means a dosage or amount sufficient toproduce a desired result. The desired result may comprise an objectiveor subjective improvement in the recipient of the dosage or amount.

A “prophylactic treatment” is a treatment administered to a subject whodoes not display signs or symptoms of a disease, pathology, or medicaldisorder, or displays only early signs or symptoms of a disease,pathology, or disorder, such that treatment is administered for thepurpose of diminishing, preventing, or decreasing the risk of developingthe disease, pathology, or medical disorder. A prophylactic treatmentfunctions as a preventative treatment against a disease or disorder. A“prophylactic activity” is an activity of an agent, such as a nucleicacid, vector, gene, polypeptide, protein, substance, or compositionthereof that, when administered to a subject who does not display signsor symptoms of pathology, disease or disorder, or who displays onlyearly signs or symptoms of pathology, disease, or disorder, diminishes,prevents, or decreases the risk of the subject developing a pathology,disease, or disorder. A “prophylactically useful” agent or compound(e.g., nucleic acid or polypeptide) refers to an agent or compound thatis useful in diminishing, preventing, treating, or decreasingdevelopment of pathology, disease or disorder.

A “therapeutic treatment” is a treatment administered to a subject whodisplays symptoms or signs of pathology, disease, or disorder, in whichtreatment is administered to the subject for the purpose of diminishingor eliminating those signs or symptoms of pathology, disease, ordisorder. A “therapeutic activity” is an activity of an agent, such as anucleic acid, vector, gene, polypeptide, protein, substance, orcomposition thereof, that eliminates or diminishes signs or symptoms ofpathology, disease or disorder, when administered to a subject sufferingfrom such signs or symptoms. A “therapeutically useful” agent orcompound (e.g., nucleic acid or polypeptide) indicates that an agent orcompound is useful in diminishing, treating, or eliminating such signsor symptoms of a pathology, disease or disorder.

The term “gene” broadly refers to any segment of DNA associated with abiological function. Genes include coding sequences and/or regulatorysequences required for their expression. Genes also includenon-expressed DNA nucleic acid segments that, e.g., form recognitionsequences for other proteins (e.g., promoter, enhancer, or otherregulatory regions). Genes can be obtained from a variety of sources,including cloning from a source of interest or synthesizing from knownor predicted sequence information, and may include sequences designed tohave desired parameters.

Generally, the nomenclature used hereafter and the laboratory proceduresin cell culture, molecular genetics, molecular biology, nucleic acidchemistry, and protein chemistry described below are those well knownand commonly employed by those of ordinary skill in the art. Standardtechniques, such as described in Sambrook et al., Molecular Cloning—ALaboratory Manual (2nd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1989 (hereinafter “Sambrook”) and CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc. (1994, supplemented through 1999)(hereinafter “Ausubel”), are used for recombinant nucleic acid methods,nucleic acid synthesis, cell culture methods, and transgeneincorporation, e.g., electroporation, injection, gene gun, impressingthrough the skin, and lipofection. Generally, oligonucleotide synthesisand purification steps are performed according to specifications. Thetechniques and procedures are generally performed according toconventional methods in the art and various general references which areprovided throughout this document. The procedures therein are believedto be well known to those of ordinary skill in the art and are providedfor the convenience of the reader.

As used herein, an “antibody” refers to a protein comprising one or morepolypeptides substantially or partially encoded by immunoglobulin genesor fragments of immunoglobulin genes. The term antibody is used to meanwhole antibodies and binding fragments thereof. The recognizedimmunoglobulin genes include the kappa, lambda, alpha, gamma, delta,epsilon and mu constant region genes, as well as myriad immunoglobulinvariable region genes. Light chains are classified as either kappa orlambda. Heavy chains are classified as gamma, mu, alpha, delta, orepsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA,IgD and IgE, respectively. A typical immunoglobulin (e.g., antibody)structural unit comprises a tetramer. Each tetramer is composed of twoidentical pairs of polypeptide chains, each pair having one “light”(about 25 KDa) and one “heavy” chain (about 50-70 KDa). The N-terminusof each chain defines a variable region of about 100 to 110 or moreamino acids primarily responsible for antigen recognition. The termsvariable light chain (VL) and variable heavy chain (VH) refer to theselight and heavy chains, respectively.

Antibodies also include single-armed composite monoclonal antibodies,single chain antibodies, including single chain Fv (sFv) antibodies inwhich a variable heavy and a variable light chain are joined together(directly or through a peptide linker) to form a continuous polypeptide,as well as diabodies, tribodies, and tetrabodies (Pack et al. (1995) MolBiol 246:28; Biotechnol 11:1271; and Biochemistry 31:1579). Theantibodies are, e.g., polyclonal, monoclonal, chimeric, humanized,single chain, Fab fragments, fragments produced by an Fab expressionlibrary, or the like.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

An “antigen-binding fragment” of an antibody is a peptide or polypeptidefragment of the antibody that binds an antigen. An antigen-binding siteis formed by those amino acids of the antibody that contribute to, areinvolved in, or affect the binding of the antigen. See Scott, T. A. andMercer, E. I., Concise Encyclopedia: Biochemistry and Molecular Biology(de Gruyter, 3d ed. 1997), and Watson, J. D. et al., Recombinant DNA (2ded. 1992) [hereinafter “Watson, Recombinant DNA”], each of which isincorporated herein by reference in its entirety for all purposes.

The term “screening” describes, in general, a process that identifiesoptimal molecules of the present invention, such as, e.g., polypeptidesof the invention, and related fusion polypeptides including the same,and nucleic acids encoding all such molecules. Several properties ofthese respective molecules can be used in selection and screening, forexample: an ability of a respective molecule to bind a ligand or to areceptor, to inhibit cell proliferation, to inhibit viral replication invirus-infected cells, to induce or inhibit cellular cytokine production,to alter an immune response, e.g., induce or inhibit a desired immuneresponse, in a test system or an in vitro, ex vivo or in vivoapplication. In the case of antigens, several properties of the antigencan be used in selection and screening including antigen expression,folding, stability, immunogenicity and presence of epitopes from severalrelated antigens.

“Selection” is a form of screening in which identification and physicalseparation are achieved simultaneously by, e.g., expression of aselection marker, which, in some genetic circumstances, allows cellsexpressing the marker to survive while other cells die (or vice versa).Screening markers include, for example, luciferase, beta-galactosidaseand green fluorescent protein, and the like. Selection markers includedrug and toxin resistance genes, and the like. Another mode of selectioninvolves physical sorting based on a detectable event, such as bindingof a ligand to a receptor, reaction of a substrate with an enzyme, orany other physical process which can generate a detectable signal eitherdirectly (e.g., by utilizing a chromogenic substrate or ligand) orindirectly (e.g., by reacting with a chromogenic secondary antibody).Selection by physical sorting can by accomplished by a variety ofmethods, such as by FACS in whole cell or microdroplet formats.

An “exogenous” nucleic acid,” “exogenous DNA segment,” “heterologoussequence,” or “heterologous nucleic acid,” as used herein, is one thatoriginates from a source foreign to the particular host cell, or, iffrom the same source, is modified from its original form. Thus, aheterologous gene in a host cell includes a gene that is endogenous tothe particular host cell, but has been modified. Modification of aheterologous sequence in the applications described herein typicallyoccurs through the use of recursive sequence recombination. The termsrefer to a DNA segment which is foreign or heterologous to the cell, orhomologous to the cell but in a position within the host cell nucleicacid in which the element is not ordinarily found. Exogenous DNAsegments are expressed to yield exogenous polypeptides.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form. Unless specifically limited, the term encompassesnucleic acids containing known analogues of natural nucleotides whichhave similar binding properties as the reference nucleic acid and aremetabolized in a manner similar to naturally occurring nucleotides.Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences and as wellas the sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al. (1991) NucleicAcid Res 19:5081; Ohtsuka et al. (1985) J Biol Chem 260:2605-2608;Cassol et al. (1992); Rossolini et al. (1994) Mol Cell Probes 8:91-98).The term nucleic acid is used interchangeably with gene, cDNA, and mRNAencoded by a gene.

“Nucleic acid derived from a gene” refers to a nucleic acid for whosesynthesis the gene, or a subsequence thereof, has ultimately served as atemplate. Thus, an mRNA, a cDNA reverse transcribed from an mRNA, an RNAtranscribed from that cDNA, a DNA amplified from the cDNA, an RNAtranscribed from the amplified DNA, etc., are all derived from the geneand detection of such derived products is indicative of the presenceand/or abundance of the original gene and/or gene transcript in asample.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For instance, apromoter or enhancer is operably linked to a coding sequence if itincreases the transcription of the coding sequence. Operably linkedmeans that the DNA sequences being linked are typically contiguous and,where necessary to join two protein coding regions, contiguous and inreading frame. However, since enhancers generally function whenseparated from the promoter by several kilobases and intronic sequencesmay be of variable lengths, some polynucleotide elements may be operablylinked but not contiguous.

The term “cytokine” includes, for example, interleukins, interferons,chemokines, hematopoietic growth factors, tumor necrosis factors andtransforming growth factors. In general these are low molecular weightproteins that regulate maturation, activation, proliferation, anddifferentiation of cells of the immune system.

In the present description and claims, any reference to “a” component,e.g. in the context of a non-polypeptide moiety, an amino acid residue,a substitution, a buffer, a cation, etc., is intended to refer to one ormore of such components, unless stated otherwise or unless it is clearfrom the particular context that this is not the case. For example, theexpression “a component selected from the group consisting of A, B andC” is intended to include all combinations of A, B and C, e.g., A, B, C,A+B, A+C, B+C or A+B+C. Various additional terms are defined orotherwise characterized herein.

Molecules and Methods of the Invention

Molecules of the invention (e.g., polypeptides of the invention,conjugates of the invention, and nucleic acids encoding saidpolypeptides) are useful for the treatment of diseases and conditionswhich are responsive to treatment by interferon-alpha, particularlydiseases and conditions associated with viral infection, such as, forexample, infection by HCV.

Patients with chronic HCV infection have viral loads typically in therange of 10⁴-10⁷ copies of HCV RNA/ml of serum prior to treatment. Upontreatment with IFN-alpha, viral load in these patientscharacteristically undergoes two distinct log-linear phases of decline(FIG. 1B; Neumann A. U., et al. (1998) Science 282:103-107). The initialrapid drop in viral load that occurs within the first two days ofIFN-alpha therapy is believed to be due to interferon-alpha mediatedreduction in virus production in the infected liver cells andconcomitant protection of naïve cells against infection. The rate ofviral production reaches a new steady state at about two days, at whichtime a second less rapid log-linear phase of viral clearance isobserved. This second phase of viral clearance is generally believed tobe due in part to T-cell mediated killing of infected liver cells(Neumann, et al., supra). IFN-alpha is believed to play a key role inthis biological response through the stimulation of antigen specific Tcells to differentiate into T_(H)1 cells. Furthermore, the mode ofaction of Ribavirin is believed to be due to augmentation of the T_(H)1response, and is thought to be the mechanistic basis of its efficacy incombination therapy with IFN-alpha. HCV-infected patients who arenon-responsive to interferon-alpha therapies currently in use (generallytermed “non-responders”) exhibit much shallower viral load clearanceprofiles (FIG. 1A).

Although the present invention is not intended to be limited by aparticular theory of underlying mechanism, it is proposed that antiviralactivity in surrogate assay systems (such as those described in moredetail herein) may be predictive of interferon-alpha efficacy, forexample in the first phase of viral clearance. An exemplary antiviralassay, described in the Examples section, monitors the effectiveness ofIFN-alpha in protecting against the cytopathic effect ofEncephalomyocarditis Virus (EMCV) in HuH7 human liver-derived cells, asa surrogate system for effectiveness against HCV in human liver cells.Example 2 shows antiviral activities of representative polypeptides ofthe invention in the EMCV/HuH7 antiviral activity assay. Preliminaryexperiments (data not shown) indicates that polypeptides of theinvention exhibit antiviral activity in other virus/cell systems,including EMCV in WISH human amniotic tissue-derived cells, EMCV in HeLahuman cervical carcinoma cells, Vesicular Stomatitis Virus (VSV) in HuH7cells, Vaccinia Virus (VV) in HeLa cells, Yellow Fever Virus (YFV) inHepG2 human hepatocarcinoma cells, as well as Human ImmunodeficiencyVirus (HIV) in human primary CD4+ T-cells. This suggests thatpolypeptides of the invention exhibit antiviral activity against a broadspectrum of viruses and cell types.

Other surrogate assay system for HCV replication in infected hepatocytesinclude HCV replicon systems, as described, for example, by Lohmann V.,et al., (1999) Science 285(5424):285-3; Randall G. and Rice C. M. (2001)Curr Opin Infect Dis 14(6):743-7; and Bartenschlager, R. (2002) NatureReviews/Drug Discovery 1:911. An example of a useful in vivo system formonitoring HCV antiviral efficacy is a chimeric human liver SCID mouse,as described by Mercer, et al. (2001) Nature Medicine 7(8):927-933.

It is furthermore proposed, without being limited by theory, thatenhancement of T_(H)1 differentiation and/or suppression of T_(H)2differentiation by IFN-alpha may be a contributing factor tointerferon-alpha efficacy, for example, in the second phase of viralclearance. According to this theory, evolved IFN-alphas with increasedpotency in these biological activities (i.e., enhancement of T_(H)1differentiation and/or suppression of T_(H)2 differentiation) would bepredicted to have increased efficacy relative to, for example, currentlyapproved therapeutic interferon-alpha molecules administered at the samedosage. An exemplary assay, described in the Examples section herein,monitors the enhancement of T_(H)1 differentiation and/or suppression ofT_(H)2 differentiation by IFN-alpha on naïve T_(H)0 cells, by measuringproduction of cytokines associated with the T_(H)1-phenotype (e.g.,IFN-gamma) and/or the T_(H)2-phenotype (e.g., IL-5, IL4) via ELISA orvia intracellular staining and FACS sorting.

The therapeutic efficacy of IFN-alpha molecules tends to be diminishedin part due to dose-limiting toxicities, e.g. thrombocytopenia andneutropenia. Although the present invention is not intended to belimited by a particular theory of underlying mechanism, it is proposedthat such toxicity may be associated with anti-proliferative effects ofIFN-alpha on platelet and neutrophil precursors, and thatantiproliferative activity in surrogate assay systems (such as thosedescribed herein) may be predictive of the relative toxicity of aninterferon-alpha molecule. Thus, dose-limiting toxicities associatedwith IFN-alpha therapy may be diminished in IFN-alpha molecules thatexhibit reduced antiproliferative activity relative to, for example,currently approved therapeutic interferon-alpha molecules, such asROFERON®-A (Interferon alfa-2a, recombinant; Hoffmann-La Roche Inc.),INTRON® A (Interferon alfa-2b, recombinant; Schering Corporation), andINFERGEN® (interferon alfacon-1; InterMune, Inc.). An exemplaryantiproliferative activity assay, described in the Examples sectionherein, monitors the effect of IFN-alpha on the proliferation of humanDaudi lymphoid cells. Alternatively, or in addition, dose-limitingtoxicities may be reduced as a result of administering moretherapeutically active molecules, which would permit dosing in lowerconcentrations or at lower frequency than currently approved molecules.

It is an object of the invention to provide novel interferon-alphapolypeptides, and nucleic acids which encode the polypeptides.Polypeptides of the invention are useful for the treatment of diseasesand disorders which are responsive to treatment by interferon-alpha,particularly diseases associated with viral infection, such as, forexample, infection by HCV. Some polypeptides of the invention exhibit aninterferon-alpha activity, such as, for example, antiviral activity,antiproliferative activity, and/or T_(H)1 differentiation activity. Somepolypeptides of the invention exhibit one or more of the followingproperties: increased or decreased antiviral activity compared to areference IFN-alpha polypeptide; increased or decreased T_(H)1differentiation activity compared to a reference IFN-alpha polypeptide;increased or decreased antiproliferative activity compared to areference IFN-alpha polypeptide. The reference IFN-alpha polypeptide maycomprise a sequence of a non-naturally occurring interferon-alpha, suchas IFN-alpha Con1 (SEQ ID NO:43), or may comprise a sequence of anaturally-occurring (i.e., wild-type) interferon-alpha polypeptide.Examples of sequences of naturally occurring interferon-alphapolypeptides include sequences of human IFN-alpha polypeptides, such as,for example, huIFN-alpha 2b (SEQ ID NO:32), huIFN-alpha 2a (SEQ ID NO:32with position 23=Lys), huIFN-alpha 2c (SEQ ID NO:32 with position34=Arg), huIFN-alpha 8b (SEQ ID NO:33), huIFN-alpha 8a (SEQ ID NO:33with positions 98=Val, 99=Leu, 100=Cys, and 101=Asp), huIFN-alpha 8c(SEQ ID NO:33 with position 161=Asp and amino acids at positions 162-166deleted), huIFN-alpha 14a (SEQ ID NO:39), huIFN-alpha 14C (SEQ ID NO:39with position 152=Leu), or a sequence of any other naturally occurringhuman interferon alpha polypeptide, such as those shown in FIGS. 2 and 4herein (SEQ ID NOs:31-42) and/or listed in Allen G. and Diaz M. O.(1996), supra.

In another aspect, the invention provides interferon-alpha polypeptideswhich exhibit enhanced efficacy in clearing a virus from virus-infectedcells, compared to a reference interferon-alpha molecule, such as onecurrently employed as a therapeutic (such as, for example, ROFERON-A,INTRON A, or INFERGEN). Exemplary viruses include, but are not limitedto, viruses of the Flaviviridae family, such as, for example, HepatitisC Virus, Yellow Fever Virus, West Nile Virus, Japanese EncephalitisVirus, Dengue Virus, and Bovine Viral Diarrhea Virus; viruses of theHepadnaviridae family, such as, for example, Hepatitis B Virus; virusesof the Picornaviridae family, such as, for example, EncephalomyocarditisVirus, Human Rhinovirus, and Hepatitis A Virus; viruses of theRetroviridae family, such as, for example, Human Immunodeficiency Virus,Simian Immunodeficiency Virus, Human T-Lymphotropic Virus, and RousSarcoma Virus; viruses of the Coronaviridae family, such as, forexample, SARS coronavirus; viruses of the Rhabdoviridae family, such as,for example, Rabies Virus and Vesicular Stomatitis Virus, viruses of theParamyxoviridae family, such as, for example, Respiratory SyncytialVirus and Parainfluenza Virus, viruses of the Papillomaviridae family,such as, for example, Human Papillomavirus, and viruses of theHerpesviridae family, such as, for example, Herpes Simplex Virus. Suchenhanced efficacy may arise from enhanced antiviral activity, enhancedT_(H)1-differentiation activity, or both, relative to the referencemolecule. For example, some interferon-alpha polypeptides of theinvention may be particularly useful in clearing viruses or viralstrains that show poor response to treatment with interferon-alphamolecules currently in use, such as, for example, Genotype 1 of HCV.

Some polypeptides of the invention exhibit an increased ratio of(antiviral activity/antiproliferative activity) compared to thereference IFN-alpha molecule, and/or an increased ratio of (T_(H)1differentiation activity/antiproliferative activity) compared to thereference IFN-alpha molecule. Polypeptides exhibiting such propertiesmay be particularly effective in treatment of viral infections, such as,for example, infection by a virus listed above. Some such polypeptidesmay, for example, provide enhanced therapeutic efficacy overcurrently-approved interferon-alpha molecules in the treatment of HCV,in one or both phases of the biphasic viral clearance profile, and/ormay exhibit reduced toxicity. Some such polypeptides may provideenhanced therapeutic efficacy over currently-approved interferon-alphamolecules in the treatment of Genotype 1 HCV.

It is another object of the invention to provide conjugates, suchconjugates comprising one or more non-polypeptide moiety linked to apolypeptide of the invention, which conjugate exhibits aninterferon-alpha activity (such as one or more of the activities listedabove), and which optionally exhibits other desirable properties, suchas increased serum half-life and/or functional in vivo half-life, and/ordecreased antigenicity, compared to the non-conjugated polypeptide. Somesuch conjugates may exhibit enhanced efficacy in clearing a virus fromcells infected with the virus, compared to a reference interferon-alphamolecule, such as an interferon-alpha conjugate currently employed as atherapeutic (such as, for example, PEGASYS® (Peginterferon alfa-2a;Hoffmann-La Roche, Inc.) or PEG-INTRON® (peginterferon alfa-2b; ScheringCorporation). Exemplary viruses include, but are not limited to, virusesof the Flaviviridae family, such as, for example, Hepatitis C Virus,Yellow Fever Virus, West Nile Virus, Japanese Encephalitis Virus, DengueVirus, and Bovine Viral Diarrhea Virus; viruses of the Hepadnaviridaefamily, such as, for example, Hepatitis B Virus; viruses of thePicornaviridae family, such as, for example, Encephalomyocarditis Virus,Human Rhinovirus, and Hepatitis A Virus; viruses of the Retroviridaefamily, such as, for example, Human Immunodeficiency Virus, SimianImmunodeficiency Virus, Human T-Lymphotropic Virus, and Rous SarcomaVirus; viruses of the Coronaviridae family, such as, for example, SARScoronavirus; viruses of the Rhabdoviridae family, such as, for example,Rabies Virus and Vesicular Stomatitis Virus, viruses of theParamyxoviridae family, such as, for example, Respiratory SyncytialVirus and Parainfluenza Virus, viruses of the Papillomaviridae family,such as, for example, Human Papillomavirus, and viruses of theHerpesviridae family, such as, for example, Herpes Simplex Virus. Suchenhanced efficacy may arise from enhanced antiviral activity, enhancedT_(H)1-differentiation activity, or both, relative to the referencemolecule. For example, some interferon-alpha conjugates of the inventionmay be particularly useful in clearing viruses or viral strains thatshow poor response to treatment with interferon-alpha moleculescurrently in use, such as, for example, Genotype 1 of HCV.

Some conjugates of the invention exhibit an increased ratio of(antiviral activity/antiproliferative activity) compared to thereference IFN-alpha molecule, and/or an increased ratio of (T_(H)1differentiation activity/antiproliferative activity) compared to thereference IFN-alpha molecule. Conjugates exhibiting such properties maybe particularly effective in treatment of viral infections, such asinfection by a virus listed above, such as, for example, HCV. Some suchconjugates may, for example, provide enhanced therapeutic efficacy overcurrently-approved interferon-alpha molecules in the treatment of HCV,in one or both phases of the biphasic viral clearance profile, and/ormay exhibit reduced toxicity. Some such conjugates may provide enhancedtherapeutic efficacy over currently-approved interferon-alpha moleculesin the treatment of Genotype 1 HCV.

It is another object of the invention to provide a method of inhibitingviral replication in virus-infected cells, the method comprisingadministering to the virus-infected cells a polypeptide or conjugate ofthe invention in an amount effective to inhibit viral replication insaid cells. The invention also provides a method of reducing the numberof copies of a virus in virus-infected cells, comprising administeringto the virus-infected cells a polypeptide or conjugate of the inventionin an amount effective to reduce the number of copies of the virus insaid cells. The virus may, for example, be a virus of the Flaviviridaefamily, such as, for example, Hepatitis C Virus, Yellow Fever Virus,West Nile Virus, Japanese Encephalitis Virus, Dengue Virus, or BovineViral Diarrhea Virus; a virus of the Hepadnaviridae family, such as, forexample, Hepatitis B Virus; a virus of the Picornaviridae family, suchas, for example, Encephalomyocarditis Virus, Human Rhinovirus, orHepatitis A Virus; a virus of the Retroviridae family, such as, forexample, Human Immunodeficiency Virus, Simian Immunodeficiency Virus,Human T-Lymphotropic Virus, or Rous Sarcoma Virus; a virus of theCoronaviridae family, such as, for example, SARS coronavirus; a virus ofthe Rhabdoviridae family, such as, for example, Rabies Virus orVesicular Stomatitis Virus, a virus of the Paramyxoviridae family, suchas, for example, Respiratory Syncytial Virus or Parainfluenza Virus, avirus of the Papillomaviridae family, such as, for example, HumanPapillomavirus, or a virus of the Herpesviridae family, such as, forexample, Herpes Simplex Virus. The virus may for example be an RNAvirus, such as HCV, a DNA virus, such as HBV, or a retrovirus, such asHIV. The cells may be in culture or otherwise isolated from a mammal(i.e., in vitro or ex vivo), or may be in vivo, e.g., in a mammal (e.g.such as a SCID mouse model as described by Mercer, et al. (2001) NatureMedicine. 7(8): 927-933), in a primate, or in man.

The invention also provides a method of enhancing T_(H)1 differentiationof T_(H)0 cells, comprising administering to a population comprisingT_(H)0 cells a polypeptide or conjugate of the invention in an amounteffective to increase the production of a cytokine associated with theT_(H)1-phenotype (e.g., IFN-gamma) and/or decrease the production of acytokine associated with the T_(H)2-phenotype (e.g., IL-4 or IL-5) insaid population. The population may be in culture or otherwise isolatedfrom a mammal (i.e., in vitro or ex vivo), or may be in vivo, e.g., in amammal, in a primate, or in man.

The invention also provides a method of inhibiting proliferation of acell population, comprising contacting the cell population with apolypeptide or conjugate of the invention in an amount effective todecrease proliferation of the cell population. The cell population maybe in culture or otherwise isolated from a mammal (i.e., in vitro or exvivo), or may be in vivo, e.g., in a mammal, a primate, or man.

These and other objects of the invention are discussed in more detailbelow.

Polypeptides of the Invention

The invention provides novel interferon-alpha polypeptides, collectivelyreferred to herein as “polypeptides of the invention”. The term“polypeptide(s) of the invention” is intended throughout to includevariants of the polypeptide sequences disclosed herein. Also included inthis invention are fusion proteins comprising polypeptides of theinvention, and conjugates comprising polypeptides of the invention.

Fragments of various interferon-alpha coding sequences were recursivelyrecombined to form libraries comprising recombinant polynucleotides,from which some polypeptides of the invention were discovered. Methodsfor obtaining libraries of recombinant polynucleotides and/or forobtaining diversity in nucleic acids used as the substrates forrecursive sequence recombination are also described infra.

Exemplary polypeptides of the invention include polypeptides comprisingsequences identified herein as SEQ ID NOs:1-15, such as SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:15, encoded by nucleicacids identified herein as SEQ ID NOs:16-30, such as SEQ ID NO:16, SEQID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30. Polypeptides of theinvention also include those comprising sequences identified herein asSEQ ID NOs:44-104, such as SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ IDNO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ IDNO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ IDNO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ IDNO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ IDNO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ IDNO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ IDNO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ IDNO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ IDNO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ IDNO: 102, SEQ ID NO:103, and SEQ ID NO:104. Some such polypeptidesfurther comprise an additional amino acid, such as a methionine, addedto the N-terminus. The invention also provides fusion proteins andconjugates comprising these polypeptides, and isolated or recombinantnucleic acids encoding these polypeptides.

The invention also includes polypeptides comprising sequences whichdiffer in 0-16 amino acid positions (such as in 0, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid positions), e.g. in 0-16positions, 0-15 positions, 0-14 positions, 0-13 positions, 0-12positions, 0-11 positions, 0-10 positions, 0-9 positions, 0-8 positions,0-7 positions, 0-6 positions, 0-5 positions, 0-4 positions, 0-3positions, 0-2 positions, or 0-1 positions, from any one of SEQ IDNOs:1-15 and SEQ ID NOs:44-104, such as, one of SEQ ID NOs:1-15, 47, and53 (for example, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12,SEQ ID NO:47, or SEQ ID NO:53). In some instances, the polypeptideexhibits an interferon-alpha activity (e.g., antiviral activity, T_(H)1differentiation activity, and/or antiproliferative activity). Some suchpolypeptides further comprise an additional amino acid, such as amethionine, added to the N-terminus. The invention also provides fusionproteins and conjugates comprising these polypeptides, and isolated orrecombinant nucleic acids encoding these polypeptides.

In some instances, the sequence of the polypeptide of the inventioncomprises a substitution of an amino acid for a different amino acid atone or more positions, including, but not limited to, positions 47, 51,52, 53, 54, 55, 56, 57, 58, 60, 61, 64, 69, 71, 72, 75, 76, 77, 78, 79,80, 83, 84, 85, 86, 87, 90, 93, 133, 140, 154, 160, 161, and 162,relative to any one of SEQ ID NOs:1-15, 47, and 53, such as, forexample, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ IDNO:47, or SEQ ID NO:53. In some instances, the polypeptide sequencecomprises one or more of: His or Gln at position 47; Val, Ala or Thr atposition 51; Gln, Pro or Glu at position 52; Ala or Thr at position 53;Phe, Ser, or Pro at position 55; Leu, Val or Ala at position 56; Phe orLeu at position 57; Tyr or His at position 58; Met, Leu or Val atposition 60; Met or Ile at position 61; Thr or Ile at position 64; Seror Thr at position 69; Lys or Glu at position 71; Asn or Asp at position72; Ala or Val at position 75; Ala or Thr at position 76; Trp or Leu atposition 77; Asp or Glu at position 78; Glu or Gln at position 79; Thr,Asp, Ser, or Arg at position 80; Glu or Asp at position 83; Lys or Gluat position 84; Phe or Leu at position 85; Tyr, Cys or Ser at position86; Ile or Thr at position 87; Phe, Tyr, Asp or Asn at position 90; Metor Leu at position 93; Lys or Glu at position 133; Ser or Ala atposition 140; Phe or Leu at position 154; Lys or Glu at position 160;Arg or Ser at position 161; and Arg or Ser at position 162; the positionnumbering relative to that of SEQ ID NO:1. The invention also providesfusion proteins and conjugates comprising these polypeptides, andisolated or recombinant nucleic acids encoding these polypeptides.

Some polypeptides of the invention comprise a substitution at a positionwhich in a parent molecule is predicted to contain an amino acid residuethat is exposed to the surface of the molecule, e.g., that is calculatedto have at least 25%, such as at least 50% of its side chain exposed tothe surface. Some such polypeptides of the invention comprise asubstitution of an amino acid for a different amino acid at one or morepositions including, but not limited to, the following positions whichcontain amino acid residues having more than 25% fractional AccessibleSurface Area (ASA): positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 16,19, 20, 22, 23, 24, 25, 26, 27, 28, 30, 31, 33, 34, 35, 37, 40, 41, 42,44, 46, 47, 49, 50, 51, 52, 59, 62, 63, 66, 69, 70, 71, 72, 74, 75, 78,79, 80, 81, 83, 84, 87, 90, 91, 94, 95, 97, 98, 100, 101, 102, 103, 104,105, 107, 108, 109, 110, 111, 112, 113, 114, 115, 118, 121, 122, 125,126, 128, 129, 132, 133, 134, 135, 136, 137, 138, 139, 146, 149, 150,153, 154, 157, 159, 160, 161, 162, 163, 164, 165, and 166, relative toany one of SEQ ID NOs:1-15, 47, and 53 (for example, SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53). Somesuch polypeptides of the invention comprise a substitution of an aminoacid for a different amino acid at one or more positions including, butnot limited to, the following positions which contain amino acidresidues having more than 50% fractional ASA: 2, 3, 4, 5, 6, 7, 8, 9,12, 13, 16, 19, 25, 27, 28, 31, 33, 34, 35, 37, 41, 44, 46, 47, 49, 50,66, 71, 75, 78, 79, 80, 83, 84, 87, 90, 91, 94, 95, 101, 102, 103, 105,107, 108, 109, 110, 111, 114, 115, 118, 121, 122, 125, 126, 129, 132,133, 135, 138, 150, 160, 162, 163, 164, 165, and 166, relative to anyone of SEQ ID NOs:1-15, 47, and 53 (for example, SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53).

Some polypeptides of the invention comprise one or more of the followingsubstitutions which introduce a cysteine residue into a position whichhas more than 25% fractional ASA: D2C, L3C, P4C, Q5C, T6C, H7C, S8C,L9C, G10C, R12C, R13C, M16C, A19C, Q20C, R22C, R23C, 124C, S25C, L26C,F27C, S28C, L30C, K31C, R33C, H34C, D35C, R37C, Q40C, E41C, E42C, D44C,N46C, H47C, Q49C, K50C, V51C, Q52C, E59C, Q62C, Q63C, N66C, S69C, T70C,K71C, N72C, S74C, A75C, D78C, E79C, T80C, L81C, E83C, K84C, 187C, F90C,Q91C, N94C, D95C, E97C, A98C, V100C, M101C, Q102C, E103C, V104C, G105C,E107C, E108C, T109C, P110C, L111C, M112C, N113C, V114C, D115C, L118C,R121C, K122C, Q125C, R126C, T128C, L129C, T132C, K133C, K134C, K135C,Y136C, S137C, P138C, A146C, M149C, R150C, S153C, F154C, N157C, Q159C,K160C, R161C, L162C, R163V, R164C, K165C and E166C (or equivalentposition relative to SEQ ID NO:1), and combinations thereof.

Some polypeptides of the invention comprise one or more of the followingsubstitutions which introduce a lysine residue into a position which hasmore than 25% fractional ASA: D2K, L3K, P4K, Q5K, T6K, H7K, S8K, L9K,G10K, R12K, R13K, M16K, A19K, Q20K, R22K, R23K, I24K, S25K, L26K, F27K,S28K, L30K, R33K, H34K, D35K, R37K, Q40K, E41K, E42K, D44K, N46K, H47K,Q49K, V51K, Q52K, E59K, Q62K, Q63K, N66K, S69K, T70K, N72K, S74K, A75K,D78K, E79K, T80K, L81K, E83K, 187K, F90K, Q91K, N94K, D95K, E97K, A98K,V100K, M101K, Q102K, E103K, V104K, G105K, E107K, E108K, T109K, P110K,L111K, M112K, N113K, V114K, D115K, L118K, R121K, Q125K, R126K, T128K,L129K, T132K, Y136K, S137K, P138K, A146K, M149K, R150K, S153K, F154K,N157K, Q159K, R161K, L162K, R163K, R164K, and E166K (or equivalentposition relative to SEQ ID NO:1), and combinations thereof.

Some polypeptides of the invention comprise a substitution of an aminoacid residue for a different amino acid residue, or a deletion of anamino acid residue, which removes one or more lysines, e.g., K31, K50,K71, K84, K122, K133, K134, K135, K160, and/or K165 (relative to SEQ IDNO:1) from any polypeptide of the invention, such as one of SEQ IDNOs:1-15 and SEQ ID NOs:44-104, such as, for example, one of SEQ IDNOs:1-15, 47, and 53 (for example, SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53). The one or morelysine residue(s) to be removed may be substituted with any other aminoacid, may be substituted with an Arg (R) or Gln (O), or may be deleted.Some such polypeptides comprise the substitutions K31R+K122R;K31R+K133R; K122R+K133R; or K31R+K122R+K133R. Other exemplarysubstitutions include K71E; K84E; K133E/G; and K160E.

Some polypeptides of the invention comprise a substitution or a deletionwhich removes one or more histidines, e.g., H7, H11, H34, and/or H47(relative to SEQ ID NO:1) from any polypeptide of the invention, such asone of SEQ ID NOs:1-15 and SEQ ID NOs:44-104, such as, for example, oneof SEQ ID NOs:1-15, 47, and 53 (for example, SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53). The one ormore histidine residue(s) to be removed may be substituted with anyother amino acid, may be substituted with an Arg (R) or Gln (Q), or maybe deleted. Some such polypeptides comprise the substitutions H34Q;H47Q; or H34Q+H47Q.

Some polypeptides of the invention comprise a substitution of an aminoacid for a different amino acid, a deletion of an amino acid, or aninsertion of an amino acid, which removes or otherwise disrupts thespatial arrangement of the N-linked glycosylation site N72 S73 S74(relative to SEQ ID NO:1). Removal of this site may be accomplished in anumber of ways, for example by deletion of N72 or substitution of N72for a different amino acid, substitution of Ser73 with Pro, substitutionof Ser74 for an amino acid other than Ser, Thr, or Cys, or insertion ofan amino acid residue other than Ser, Thr, or Cys between positions 73and 74. For example, some such polypeptides comprise the substitutionN72D.

Some polypeptides of the invention comprise one or more amino acidsubstitution, deletion or insertion which removes one or more basicresidues or one or more pairs of basic residues (such as, Arg-Arg,Arg-Lys, Lys-Arg, Lys-Lys) in order to, for example, minimize thepresence of potential protease-sensitive sites, or in some instances toremove sites potentially reactive towards amine-reactive conjugation(e.g. PEGylation) reagents. For example, removal of dibasic sequencesnear the C-terminus may be accomplished by removal of one or more ofLys160, Arg161, Arg163, Arg164, and Lys165 (relative to SEQ ID NO:1).The one or more Lys or Arg to be removed may for example be deleted, orsubstituted with any amino acid other than Lys or Arg. Some suchpolypeptides of the invention comprise a substitution of one or more ofLys160, Arg161, and Arg164 for an amino acid other than Lys or Arg, suchas, for example, one or more of the substitutions Lys160Glu;Arg161Ser/Cys; and Arg164Ser/Cys. Some such polypeptides alternativelyor in addition comprise a deletion of one or more of Lys 160, Arg161,Arg163, Arg164, and Lys165, which may be via individual deletions (e.g.,K165*) or in groups of more than one, including via C-terminaltruncation (e.g., K165*-E166*).

Other modifications contemplated for polypeptides of the inventioninclude those described below and in the section entitled“INTERFERON-ALPHA CONJUGATES”.

It is to be understood that while the examples and modifications to theparent polypeptide are generally provided herein relative to thesequence SEQ ID NO:1 (or relative to some other specified sequence), thedisclosed modifications may also be made in equivalent amino acidpositions of any of the other polypeptides of the invention (includingSEQ ID NOs:2-15 and SEQ ID NOs:44-104 and variants thereof) describedherein. Thus, as an example, the substitution H47C relative to SEQ IDNO:1 is understood to correspond to Q47C in SEQ ID NO:5, and so on.

The following tables provide sequences of some interferon-alphapolypeptides of the invention. For clarity, the sequences are shownrelative to SEQ ID NO:3 (Table 1) or SEQ ID NO:12 (Table 2). Some suchpolypeptides exhibit an interferon-alpha activity, such as antiviralactivity, T_(H)1 differentiation activity, and/or antiproliferativeactivity.

TABLE 1 Polypeptide Sequence (relative to SEQ ID NO:3) Clone name(s) SEQID SEQ ID NO:3 B9x11 SEQ ID NO:1 +E133K, A140S SEQ ID NO:3 B9x12 SEQ IDNO:2 +H47Q, E133K, A140S SEQ ID NO:3 B9x14, SEQ ID NO:3 B9x14CHO2 SEQ IDNO:3 B9x15 SEQ ID NO:4 +H47Q, V51T, F55S, L56V, Y58H, E133K, A140S SEQID NO:3 B9x16 SEQ ID NO:5 +H47Q SEQ ID NO:3 B9x17 SEQ ID NO:6 +V51T,F55S, L56V, Y58H SEQ ID NO:3 B9x18 SEQ ID NO:7 +H47Q, V51T, F55S, L56V,Y58H SEQ ID NO:3 B9x14C2a SEQ ID NO:44 +F154L, K160E, R161S, R164S SEQID NO:3 B9x14CHO1 SEQ ID NO:45 +E166EC SEQ ID NO:3 B9x14CHO3 SEQ IDNO:46 +N72D SEQ ID NO:3 B9x14CHO4, SEQ ID NO:47 +N72D, K160E, R161S,R164S B9x14EC4 SEQ ID NO:3 B9x14CHO5, SEQ ID NO:48 +N72D, K160E, R161C,R164S B9x14EC5 SEQ ID NO:3 B9x14CHO6, SEQ ID NO:49 +N72D, K160E, R161S,R164C B9x14EC3 SEQ ID NO:3 14Ep01 SEQ ID NO:50 +H47Q, V51A, F55S, L56V,F57L, Y58H, N72D, F154L, K160E, R161S, R164S SEQ ID NO:3 14Ep02 SEQ IDNO:51 +M61I, N72D, E83D, M93L, M101T, T109I, P110A, V114E, F154L, K160E,R161S, R164S SEQ ID NO:3 14Ep03 SEQ ID NO:52 +N72D, M101I, F154L, K160E,R161S, R164S SEQ ID NO:3 14Ep04 SEQ ID NO:53 +N72D, F154L, K160E, R161S,R164S SEQ ID NO:3 14Ep05 SEQ ID NO:54 +H47Q, V51A, F55S, L56V, F57L,Y58H, N72D, M101I, F154L, K160E, R161S, R164S SEQ ID NO:3 14EF SEQ IDNO:55 +H47Q, V51A, F55S, L56V, F57L, Y58H, M61I, N72D, E83D, M93L,M101T, T109I, P110A, V114E, F154L, K160E, R161S, R164S SEQ ID NO:3B9x14Ep04C31 SEQ ID NO:56 +K31C, N72D, F154L, K160E, R161S, R164S SEQ IDNO:3 B9x14CHO4C31 SEQ ID NO:57 +K31C, N72D, K160E, R161S, R164S SEQ IDNO:3 B9x14CHO4C46 SEQ ID NO:58 +N46C, N72D, K160E, R161S, R164S SEQ IDNO:3 B9x14CHO4C71 SEQ ID NO:59 +K71C, N72D, K160E, R161S, R164S SEQ IDNO:3 B9x14CHO4C75 SEQ ID NO:60 +N72D, A75C, K160E, R161S, R164S SEQ IDNO:3 B9x14 CHO4C79 SEQ ID NO:61 +N72D, E79C, K160E, R161S, R164S SEQ IDNO:3 B9x14CHO4C107 SEQ ID NO:62 +N72D, E107C, K160E, R161S, R164S SEQ IDNO:3 B9x14 CHO4C122 SEQ ID NO:63 +N72D, K122C, K160E, R161S, R164S SEQID NO:3 B9x14CHO4C134 SEQ ID NO:64 +N72D, K134C, K160E, R161S, R164S SEQID NO:3 B9x14Ep04 SEQ ID NO:65 +N72D, F154L, K160E, R161*- Δ161-166E166* SEQ ID NO:3 B9x14Ep04 SEQ ID NO:66 +N72D, F154L, K160E, R161S,Δ165-166 R164S, K165*-E166* SEQ ID NO:3 B9x14Ep04Δ1-4 SEQ ID NO:67+C1*-P4*, D44*, N72D, F154L, D44*Δ165-166 K160E, R161S, R164S, K165*-E166* SEQ ID NO:3 B9x14CHO4NP1 SEQ ID NO:68 +H34Q, N72D, K160E, R161S,R164S SEQ ID NO:3 B9x14 CHO4NP2 SEQ ID NO:69 +H34Q, H47Q, N72D, K160E,R161S, R164S SEQ ID NO:3 B9x14CHO8 SEQ ID NO:70 +K31R, N72D, K160E,R161S, R164S SEQ ID NO:3 B9x14CHO9 SEQ ID NO:71 +K50R, N72D, K160E,R161S, R164S SEQ ID NO:3 B9x14CHO10 SEQ ID NO:72 +K71R, N72D, K160E,R161S, R164S SEQ ID NO:3 B9x14CHO11 SEQ ID NO:73 +N72D, K84R, K160E,R161S, R164S SEQ ID NO:3 B9x14CHO12 SEQ ID NO:74 +N72D, K122R, K160E,R161S, R164S SEQ ID NO:3 B9x14CHO13 SEQ ID NO:75 +N72D, K134R, K160E,R161S, R164S SEQ ID NO:3 B9x14CHO14 SEQ ID NO:76 +N72D, K135R, K160E,R161S, R164S SEQ ID NO:3 B9x14CHO15 SEQ ID NO:77 +N72D, K160E, R161S,R164S, K165R SEQ ID NO:3 B9x14CHO16 SEQ ID NO:78 +N72D, K122R, K135R,K160E, R161S, R164S SEQ ID NO:3 B9x14CHO17 SEQ ID NO:79 +K31R, N72D,K135R, K160E, R161S, R164S SEQ ID NO:3 B9x14CHO18 SEQ ID NO:80 +K31R,N72D, K122R, K160E, R161S, R164S SEQ ID NO:3 B9x14CHO18NP2 SEQ ID NO:81+K31R, H34Q, H47Q, N72D, K122R, K160E, R161S, R164S SEQ ID NO:3B9x14CHO18NP2 SEQ ID NO:82 +K31R, H34Q, H47Q, N72D, Δ165-166 K122R,K160E, R161S, R164S, K165*-E166*

TABLE 2 Polypeptide Sequence (relative to SEQ ID NO:12) Clone name(s)SEQ ID SEQ ID NO:12 B9X21 SEQ ID NO:8 +H47Q, V51T, F55S, L56V, Y58H SEQID NO:12 B9X22 SEQ ID NO:9 +V51T, F55S, L56V, Y58H SEQ ID NO:12 B9X23SEQ ID NO:10 +H47Q SEQ ID NO:12 B9X24 SEQ ID NO:11 +H47Q, V51T, F55S,L56V, Y58H, E133K, A140S SEQ ID NO:12 B9X25 SEQ ID NO:12 SEQ ID NO:12B9X26 SEQ ID NO:13 +V51T, F55S, L56V, Y58H, E133K, A140S SEQ ID NO:12B9X27 SEQ ID NO:14 +H47Q, E133K, A140S SEQ ID NO:12 B9X28 SEQ ID NO:15+E133K, A140S SEQ ID NO:12 B9x25CHO1 SEQ ID NO:83 +N72D SEQ ID NO:12B9x25CHO2, SEQ ID NO:84 +N72D, F154L, K160E, R161S, 25Ep09, R164SB9x25EC1 SEQ ID NO:12 B9x25CHO3, SEQ ID NO:85 +N72D, F154L, K160E,R161C, B9x25EC2 R164S SEQ ID NO:12 B9x25CHO4, SEQ ID NO:86 +N72D, F154L,K160E, R161S, B9x25EC3 R164C SEQ ID NO:12 25Ep01 SEQ ID NO:87 +H47Q,V51T, F55S, L56V, Y58H, N72D SEQ ID NO:12 25Ep02 SEQ ID NO:88 +L17I,R22G, H47Q, V51T, F55S, L56V, Y58H, N72D, F154L, K160E, R161S, R164S SEQID NO:12 25Ep03 SEQ ID NO:89 +D2N, P4S, S10N, H47Q, V51T, F55S, L56V,Y58H, N72D, F154L, K160E, R161S, R164S SEQ ID NO:12 25Ep04 SEQ ID NO:90+H47Q, V51T, F55S, L56V, Y58H, L60M, N72D, F154L, K160E, R161S, R164SSEQ ID NO:12 25Ep05 SEQ ID NO:91 +H47Q, V51T, F55S, L56V, Y58H, N72D,N95D, F154L, K160E, R161S, R164S SEQ ID NO:12 25Ep06 SEQ ID NO:92 +H47Q,V51T, F55S, L56V, Y58H, N72D, E83D, M93L, N95D, I101T, V114E, F154L,K160E, R161S, R164S SEQ ID NO:12 25Ep07 SEQ ID NO:93 +H47Q, V51T, F55S,L56V, Y58H, N72D, R125Q, F154L, K160E, R161S, R164S SEQ ID NO:12 25Ep08SEQ ID NO:94 +H47Q, V51T, F55S, L56V, Y58H, N72D, F154L, K160E, R161S,R164S SEQ ID NO:12 25Ep10 SEQ ID NO:95 +L17I, R22G, N72D, F154L, K160E,R161S, R164S SEQ ID NO:12 25Ep11 SEQ ID NO:96 +D2N, P4S, S10N, N72D,F154L, K160E, R161S, R164S SEQ ID NO:12 25Ep12 SEQ ID NO:97 +N72D,R125Q, F154L, K160E, R161S, R164S SEQ ID NO:12 25Ep13 SEQ ID NO:98+L17I, R22G, N72D, R125Q, F154L, K160E, R161S, R164S SEQ ID NO:12 25Ep14SEQ ID NO:99 +D2N, P4S, S10N, N72D, R125Q, F154L, K160E, R161S, R164SSEQ ID NO:12 25Ep15 SEQ ID NO:100 +H47Q, V51T, F55S, L56V, Y58H, N72D,F154L, K160E, R161S, R164S SEQ ID NO:12 25Ep16 SEQ ID NO:101 +L17I,R22G, H47Q, V51T, F55S, L56V, Y58H, N72D, R125Q, F154L, K160E, R161S,R164S SEQ ID NO:12 25Ep17 SEQ ID NO:102 +D2N, P4S, S10N, H47Q, V51T,F55S, L56V, Y58H, N72D, R125Q, F154L, K160E, R161S, R164S SEQ ID NO:1225EF1 SEQ ID NO:103 +L17I, R22G, H47Q, V51T, F55S, L56V, Y58H, L60M,N72D, E83D, M93L, N95D, I101T, V114E, R125Q, F154L, K160E, R161S, R164SSEQ ID NO:12 25EF2 SEQ ID NO:104 +D2N, P4S, S10N, H47Q, V51T, F55S,L56V, Y58H, L60M, N72D, E83D, M93L, N95D, I101T, V114E, R125Q, F154L,K160E, R161S, R164SVariants

In another aspect, the invention provides an isolated or recombinantpolypeptide which is a variant of a parent interferon-alpha polypeptide,the variant comprising a sequence which differs from the parentpolypeptide sequence in least one amino acid position, wherein thevariant sequence comprises one or more of His at position 47, Val atposition 51, Phe at position 55, Leu at position 56, Tyr at position 58,Lys at position 133, and at position Ser140, the position numberingrelative to that of SEQ ID NO:1. In some instances the parentinterferon-alpha polypeptide sequence is a sequence of anaturally-occurring human interferon-alpha (such as, for example,huIFN-alpha 2b (SEQ ID NO:32), huIFN-alpha 2a (SEQ ID NO:32 withposition 23=Lys), huIFN-alpha 2c (SEQ ID NO:32 with position 34=Arg),huIFN-alpha 8b (SEQ ID NO:33), huIFN-alpha 8a (SEQ ID NO:33 withpositions 98=Val, 99=Leu, 100=Cys, and 101=Asp), huIFN-alpha 8c (SEQ IDNO:33 with position 161=Asp and amino acids at positions 162-166deleted), huIFN-alpha 14a (SEQ ID NO:39), huIFN-alpha 14c (SEQ ID NO:39with position 152=Leu), or a sequence of any other naturally occurringhuman interferon alpha polypeptide, such as those shown in FIGS. 2 and 4herein (SEQ ID NOs:31-42) and/or listed in Allen G. and Diaz M. O.(1996), supra). In some instances the parent interferon-alphapolypeptide sequence is a sequence of a non-naturally occurring (i.e.,synthetic) interferon-alpha, such as IFN-alphaCon1 (SEQ ID NO:43) Insome instances, the parent polypeptide to be modified may itself be apolypeptide of the invention, such as, e.g. any one of SEQ ID NOs:1-15and SEQ ID NOs:44-104. In some instances, the variant sequence differsfrom the parent polypeptide sequence in 1-16 amino acid positions (suchas in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 aminoacid positions), e.g. in 1-14 amino acid positions, in 1-12 amino acidpositions, in 1-10 amino acid positions, in 1-8 amino acid positions, in1-6 amino acid positions, in 1-5 amino acid positions, in 1-4 amino acidpositions, in 1-3 amino acid positions, or in 1-2 amino acid positions.Some such variants exhibit an interferon-alpha activity. The inventionalso provides fusion proteins and conjugates comprising these variants,and isolated or recombinant nucleic acids encoding these variants.

Sequence Variations

As noted above, polypeptides of the present invention includepolypeptides comprising sequences which differ from any one of SEQ IDNOs:1-15 and SEQ ID NOs:44-104, such as one of SEQ ID NOs:1-15, 47, and53 (for example, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12,SEQ ID NO:47, or SEQ ID NO:53), in 0-16 amino acid positions (such as in0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acidpositions), e.g. in 0-16 positions, 0-15 positions, 0-14 positions, 0-13positions, 0-12 positions, 0-11 positions, 0-10 positions, 0-9positions, 0-8 positions, 0-7 positions, 0-6 positions, 0-5 positions,0-4 positions, 0-3 positions, 0-2 positions, or 0-1 positions. Some suchpolypeptides exhibit an interferon-alpha activity.

For example, some such polypeptides of the invention comprise a sequencehaving a length of about 150 amino acids, such as about 151, 152, 153,154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, or 165 aminoacids, corresponding to a deletion of between 1 and 16 amino acidsrelative to a parent polypeptide sequence (such as, for example, one ofSEQ ID NOs:1-15). In some instances, between 1 and 11, e.g., between 1and 10, such as between 1 and 7, e.g. between 1 and 5, such as between 1and 3 amino acids are deleted from the C-terminus, i.e. the polypeptideis C-terminally truncated compared to the parent polypeptide sequence(such as, for example, one of SEQ ID NOs: 1-15, 47, or 53) by 1-11 aminoacid residues (e.g. by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acidresidues), such as by 1-10, 1-7, e.g., by 1-5 or by 1-3 amino acidresidues. Alternatively, or in addition, some such polypeptides areN-terminally truncated compared to the parent polypeptide sequence (suchas, one of SEQ ID NOs:1-15, 47, or 53) by 1-4 amino acid residues (e.g.by 1, 2, 3, or 4 amino acid residues), e.g., 1-4, 1-3, 1-2 or 1 aminoacid residue(s) are removed from the N-terminus. Some such polypeptidesfurther comprise a methionine at the N-terminus. Some such polypeptidesexhibit an interferon-alpha activity.

As another example, some such polypeptides of the invention comprise asequence containing between 0 and 16 amino acid substitutions relativeto one of SEQ ID NOs:1-15, 47, or 53 (e.g. 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, or 16 amino acid substitutions), such as 0-14 or0-12 or 0-10 or 0-8 or 0-6 or 0-5 or 0-4 or 0-3 or 0-2 or 0-1 amino acidsubstitutions. In some instances, one or more of the amino acidsubstitutions are made according to, for example, a substitution group(such as, a conservative substitution group), such as one set forthbelow. Some such polypeptides exhibit an interferon-alpha activity.

Some polypeptides of the invention comprise a sequence comprisingbetween 0 and 16 amino acid substitutions relative to one of SEQ IDNOs:1-15 and SEQ ID NOs:44-104 (for example, SEQ ID NO:1, SEQ ID NO:3,SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53), e.g. 0, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acidsubstitutions, such as 0-14 or 0-12 or 0-10 or 0-8 or 0-6 or 0-5 or 0-4or 0-3 or 0-2 or 0-1 amino acid substitutions, where at least one ofsaid substitution(s) introduces an amino acid residue comprising anattachment group for a non-polypeptide moiety. Examples includeintroduction of one or more N-glycosylation site(s), or introduction ofone or more cysteine residue(s) or lysine residue(s), as described aboveand in the section entitled “INTERFERON-ALPHA CONJUGATES”. Some suchpolypeptides exhibit an interferon-alpha activity.

Some polypeptides of the invention comprise a sequence containingbetween 0 and 16 amino acid substitutions or deletions or insertionsrelative to one of SEQ ID NOs:1-15 and SEQ ID NOs:44-104 (for example,SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47, orSEQ ID NO:53), such as 0-14 or 0-12 or 0-10 or 0-8 or 0-6 or 0-5 or 0-4or 0-3 or 0-2 or 0-1 amino acid substitutions deletions or insertions(or a combination thereof), where at least one of said substitution ordeletion removes an amino acid residue from a parent polypeptidesequence which comprises an attachment group for a non-polypeptidemoiety or, in the case of an amino acid insertion, disrupts the spatialarrangement of residues required for such attachment group (e.g., aninsertion of an amino acid to disrupt an N-glycosylation N-X-S/T motif).Examples include removal from the parent polypeptide sequence of anN-glycosylation site, or removal of a lysine, histidine, or cysteineresidue, as described above and in the section entitled“INTERFERON-ALPHA CONJUGATES”. Some such polypeptides exhibit aninterferon-alpha activity.

As a non-limiting example, a polypeptide of the invention may have thesequence SEQ ID NO:3 or a sequence which differs from SEQ ID NO:3 in atotal of up to 16 positions (which may be a combination of amino acidsubstitutions, deletions, and/or insertions, including those describedabove). In some instances, none, some, or all of the substitutions aresubstitutions according to a substitution group defined below.

Amino acid substitutions in accordance with the invention may include,but are not limited to, one or more conservative amino acidsubstitutions. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. One example is providedin the table below (Table 3), which sets forth six exemplary groups thatcontain amino acids which may be considered “conservative substitutions”for one another.

TABLE 3 Conservative Substitution Groups 1 Alanine (A) Glycine (G)Serine (S) Threonine (T) 2 Aspartic acid (D) Glutamic acid (E) 3Asparagine (N) Glutamine (Q) 4 Arginine (R) Lysine (K) Histidine (H) 5Isoleucine (I) Leucine (L) Methionine (M) Valine (V) 6 Phenylalanine (F)Tyrosine (Y) Tryptophan (W)

Other substitution groups of amino acids can be envisioned. For example,amino acids can be grouped by similar function or chemical structure orcomposition (e.g., acidic, basic, aliphatic, aromatic,sulfur-containing). For example, an Aliphatic grouping may comprise:Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I). Othergroups containing amino acids that are considered conservativesubstitutions for one another include: Aromatic: Phenylalanine (F),Tyrosine (Y), Tryptophan (W); Sulfur-containing: Methionine (M),Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic:Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q). Seealso Creighton (1984) Proteins, W.H. Freeman and Company, for additionalgroupings of amino acids. Listing of a polypeptide sequence herein, inconjunction with the above substitution groups, provides an expresslisting of all conservatively substituted polypeptide sequences.

Percent Sequence Identity

In one aspect, the invention provides an isolated or recombinantpolypeptides each comprising a sequence having at least 90% sequenceidentity (e.g., at least about 91%, at least about 92%, at least about93%, at least about 94%, at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99% amino acidsequence identity) to any one of SEQ ID NOs: 1-15 and SEQ ID NOs:44-104,such as, for example, to one of SEQ ID NOs:1-15, 47, and 53 (forexample, SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ IDNO:47, or SEQ ID NO:53). In some instances the polypeptide exhibits aninterferon-alpha activity. In some instances, the polypeptide sequencediffers at one or more amino acid positions, e.g., in up to 16 positions(such as, 1-16 positions, 1-15 positions, 1-14 positions, 1-13positions, 1-12 positions, 1-11 positions, 1-10 positions, 1-9positions, 1-8 positions, 1-7 positions, 1-6 positions, 1-5 positions,1-4 positions, 1-3 positions, or 1-2 positions) from any one of SEQ IDNO:1-15 and 44-104, such as, for example, SEQ ID NO:1, SEQ ID NO:3, SEQID NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53. As an example,some positions which may be substituted for another amino acid inaccordance with the invention include, but are not limited to, one ormore of positions 47, 51, 52, 53, 54, 55, 56, 57, 58, 60, 61, 64, 69,71, 72, 75, 76, 77, 78, 79, 80, 83, 84, 85, 86, 87, 90, 93, 133, 140,154, 160, 161, and 162, relative to one of SEQ ID NOs:1-15 and 44-104.In some instances, the sequence comprises one or more of: His or Gln atposition 47; Val, Ala or Thr at position 51; Gln, Pro or Glu at position52; Ala or Thr at position 53; Phe, Ser, or Pro at position 55; Leu, Valor Ala at position 56; Phe or Leu at position 57; Tyr or His at position58; Met, Leu or Val at position 60; Met or Ile at position 61; Thr orIle at position 64; Ser or Thr at position 69; Lys or Glu at position71; Asn or Asp at position 72; Ala or Val at position 75; Ala or Thr atposition 76; Trp or Leu at position 77; Asp or Glu at position 78; Gluor Gln at position 79; Thr, Asp, Ser, or Arg at position 80; Glu or Aspat position 83; Lys or Glu at position 84; Phe or Leu at position 85;Tyr, Cys or Ser at position 86; Ile or Thr at position 87; Phe, Tyr, Aspor Asn at position 90; Met or Leu at position 93; Lys or Glu at position133; Ser or Ala at position 140; Phe or Leu at position 154; Lys or Gluat position 160; Arg or Ser at position 161; and Arg or Ser at position162; the position numbering relative to that of SEQ ID NO:1. Othersubstitutions contemplated in sequences of the invention are describedabove and in the section entitled “INTERFERON-ALPHA CONJUGATES”. Theinvention also provides fusion proteins comprising such polypeptides,conjugates comprising such polypeptides, and isolated or recombinantnucleic acids encoding such polypeptides.

In another aspect, the present invention provides nucleic acids havingat least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more percent sequence identity to one or more ofSEQ ID NOS:16-30. Some such nucleic acids encode polypeptides exhibitingan interferon-alpha activity as described herein.

The degree to which a sequence (polypeptide or nucleic acid) is similarto another provides an indication of similar structural and functionalproperties for the two sequences. Accordingly, in the context of thepresent invention, sequences which have a similar sequence to any givenexemplar sequence are a feature of the present invention. In particular,sequences that have percent sequence identities as defined below are afeature of the invention.

A variety of methods of determining sequence relationships can be used,including manual alignment and computer assisted sequence alignment andanalysis. A variety of computer programs for performing sequencealignments are available, or an alignment can be prepared manually byone of skill, as described below.

As noted above, the sequences of the nucleic acids and polypeptidesemployed in the subject invention need not be identical, but can besubstantially identical to the corresponding sequence of a polypeptideof the invention or nucleic acid of the invention. For example,polypeptides of the invention can be subject to various changes, such asone or more amino acid insertions, deletions, and/or substitutions,either conservative or non-conservative, including where, e.g., suchchanges might provide for certain advantages in their use, such as, intheir therapeutic or prophylactic use or administration or diagnosticapplication. The nucleic acids of the invention can also be subject tovarious changes, such as one or more substitutions of one or morenucleic acids in one or more codons such that a particular codon encodesthe same or a different amino acid, resulting in either a silentvariation (as defined herein) or non-silent variation, or one or moredeletions of one or more nucleic acids (or codons) in the sequence. Thenucleic acids can also be modified to include one or more codons thatprovide for optimum expression in an expression system (e.g., bacterialor mammalian), while, if desired, said one or more codons still encodethe same amino acid(s). Such nucleic acid changes might provide forcertain advantages in their therapeutic or prophylactic use oradministration, or diagnostic application. The nucleic acids andpolypeptides can be modified in a number of ways so long as theycomprise a sequence substantially identical (as defined below) to asequence in a respective nucleic acid or polypeptide of the invention.

The term “identical” or “identity,” in the context of two or morenucleic acid or polypeptide sequences, refers to two or more sequencesthat are the same or have a specified percentage of amino acid residuesor nucleotides that are the same, when compared and aligned for maximumsimilarity, as determined using the sequence comparison algorithmdescribed below or by visual inspection.

The “percent sequence identity” (“% identity”) of a subject sequence toa reference (i.e. query) sequence means that the subject sequence isidentical (i.e., on an amino acid-by-amino acid basis for a polypeptidesequence, or a nucleotide-by-nucleotide basis for a polynucleotidesequence) by a specified percentage to the query sequence over acomparison length.

The percent sequence identity of a subject sequence to a query sequenceis calculated as follows. First, the optimal alignment of the twosequences is determined using a sequence comparison algorithm withspecific alignment parameters. This determination of the optimalalignment may be performed using a computer, or may be manuallycalculated, as described below. Then, the two optimally alignedsequences are compared over the comparison length, and the number ofpositions in the optimal alignment at which identical residues occur inboth sequences are determined, which provides the number of matchedpositions. The number of matched positions is then divided by the totalnumber of positions of the comparison length (which, unless otherwisespecified, is the length of the query sequence), and then the result ismultiplied by 100, to yield the percent sequence identity of the subjectsequence to the query sequence.

With regard to polypeptide sequences, typically one sequence is regardedas a “query sequence” (for example, a polypeptide sequence of theinvention) to which one or more other sequences, i.e., “subjectsequence(s)” (for example, sequences present in a sequence database) arecompared. The sequence comparison algorithm uses the designatedalignment parameters to determine the optimal alignment between thequery sequence and the subject sequence(s). When comparing a querysequence against a sequence database, such as, e.g., GENBANK® (GeneticSequence Data Bank; U.S. Department of Health and Human Services) orGENESEQ® (Thomson Derwent; also available as DGENE® on STN), usuallyonly the query sequence and the alignment parameters are input into thecomputer; optimal alignments between the input query sequence and eachsubject sequence present in the database are returned, generally for upto a desired number of subject sequences.

Two polypeptide sequences are “optimally aligned” when they are alignedusing defined parameters, i.e., a defined amino acid substitutionmatrix, gap existence penalty (also termed gap open penalty), and gapextension penalty, so as to arrive at the highest similarity scorepossible for that pair of sequences. The BLOSUM62 matrix (Henikoff andHenikoff (1992) Proc. Natl. Acad. Sci. USA 89(22):10915-10919) is oftenused as a default scoring substitution matrix in polypeptide sequencealignment algorithms (such as BLASTP, described below). The gapexistence penalty is imposed for the introduction of a single amino acidgap in one of the aligned sequences, and the gap extension penalty isimposed for each residue position in the gap. Unless otherwise stated,alignment parameters employed herein are: BLOSUM62 scoring matrix, gapexistence penalty=11, and gap extension penalty=1. The alignment scoreis defined by the amino acid positions of each sequence at which thealignment begins and ends (e.g. the alignment window), and optionally bythe insertion of a gap or multiple gaps into one or both sequences, soas to arrive at the highest possible similarity score. While optimalalignment between two or more sequences can be determined manually (asdescribed below), the process is facilitated by the use of acomputer-implemented alignment algorithm such as BLAST® (NationalLibrary of Medicine), e.g., BLASTP for polypeptide sequences and BLASTNfor nucleic acid sequences, described in Altschul et al. (1997) NucleicAcids Res. 25:3389-3402, and made available to the public throughvarious sources, such as the National Center for BiotechnologyInformation (NCBI) Website. When using a computerized BLAST interface,if the option exists to use a “low complexity filter”, this optionshould be turned off (i.e., no filter).

FIG. 3 shows an alignment of a polypeptide of the invention (B9×14, SEQID NO:3) with human IFN-alpha 14a (also known as LeIF H, Goeddel et al.(1981) Nature 290:20-26; SEQ ID NO:39), which was the mostclosely-related sequence retrieved in a BLASTP search of query sequenceSEQ ID NO:3 against the GENBANK and GENESEQ databases using the BLOSUM62matrix, gap open penalty 11, gap extension penalty 1. These twosequences differ in 18 amino acid positions over a length of 166 aminoacids (i.e., SEQ ID NO:39 differs from SEQ ID NO:3 in 18 amino acidpositions); furthermore, SEQ ID NO:39 is 89% identical to SEQ ID NO:3,since ((166-18)/166)×100=89.

FIG. 5 shows an alignment of another polypeptide of the invention(B9×25, SEQ ID NO: 12) with human IFN-alpha 14a (SEQ ID NO:39) which wasthe most closely-related sequence retrieved in a BLASTP search of querysequence SEQ ID NO:12 against the GENBANK and GENESEQ databases usingthe parameters specified above. SEQ ID NO:39 differs from SEQ ID NO:12in 20 amino acid positions over a length of 166 amino acids;furthermore, SEQ ID NO:39 is 88% identical to SEQ ID NO:3, since((166-20)/166)×100=88.

The optimal alignment between two polypeptide sequences can also bedetermined by a manual calculation of the BLASTP algorithm (i.e.,without aid of a computer) using the same alignment parameters specifiedabove (matrix=BLOSUM62, gap open penalty=11, and gap extensionpenalty=1). To begin, the two sequences are initially aligned by visualinspection. An initial alignment score is then calculated as follows:for each individual position of the alignment (i.e., for each pair ofaligned residues), a numerical value is assigned according to theBLOSUM62 matrix (FIG. 6). The sum of the values assigned to each pair ofresidues in the alignment is the initial alignment score. If the twosequences being aligned are highly similar, often this initial alignmentprovides the highest possible alignment score. The alignment with thehighest possible alignment score is the optimal alignment based on thealignment parameters employed. FIG. 7A shows an example calculation ofan alignment score for two sequences, a “query” sequence, identifiedherein as residues 29-50 of SEQ ID NO:3 (upper), and a “subject”sequence, identified herein as residues 30-52 of SEQ ID NO:5 (lower).The sequences were aligned by visual inspection, and the numerical valueassigned by the BLOSUM62 matrix for each aligned pair of amino acids isshown beneath each position in the alignment (to aid in visualization,each identical pair of amino acids in the alignment is shown inboldface). In this example, this initial alignment provided the highestpossible alignment score (the sum of the values shown beneath eachaligned position); any other alignment of these two sequences, with orwithout gaps, would result in a lower alignment score.

In some instances, a higher alignment score might be obtained byintroducing one or more gaps into the alignment. Whenever a gap isintroduced into an alignment, a gap open penalty is assigned, and inaddition a gap extension penalty is assessed for each residue positionwithin that gap. Therefore, using the alignment parameters describedabove (including gap open penalty=11 and gap extension penalty=1), a gapof one residue in the alignment would correspond to a value of−(11+(1×1))=−12 assigned to the gap; a gap of three residues wouldcorrespond to a value of −(11+(3×1))=−14 assigned to the gap, and so on.This calculation is repeated for each new gap introduced into thealignment. FIGS. 7B and 7C show an example which demonstrates howintroduction of a gap into an alignment can result in a higher alignmentscore, despite the gap penalty. FIG. 7B shows an initial alignment ofresidues 29-50 of SEQ ID NO:3 (upper, query) and residues 30-50 of SEQID NO:32 (lower, subject) made by visual inspection, which results in aninitial alignment score of 67. FIG. 7C shows the effect of a one-residuegap in SEQ ID NO:32 on the alignment score; despite the gap penalty of−12, the overall alignment score of the two sequences increases to 88.In this example, the alignment shown in FIG. 7C provides the highestpossible alignment score, and is thus the optimal alignment of these twosequences; any other alignment of these two sequences (with or withoutgaps) would result in a lower alignment score.

It is to be understood that the examples of sequence alignmentcalculations described above, which use relatively short sequences, areprovided for illustrative purposes only; in practice, the alignmentparameters employed (BLOSUM62 matrix, gap open penalty=11, and gapextension penalty=1) are generally intended for polypeptide sequences 85amino acids in length or longer. The NCBI website provides the followingalignment parameters for sequences of other lengths (which are suitablefor computer-aided as well as manual alignment calculation, using thesame procedure as described above). For sequences of 50-85 amino acidsin length, optimal parameters are BLOSUM80 matrix (Henikoff andHenikoff, supra), gap open penalty=10, and gap extension penalty=1. Forsequences of 35-50 amino acids in length, optimal parameters are PAM70matrix (Dayhoff, M. O., Schwartz, R. M. & Orcutt, B. C. (1978) “A modelof evolutionary change in proteins.” In Atlas of Protein Sequence andStructure, vol. 5, suppl. 3, M. O. Dayhoff (ed.), pp. 345-352, Natl.Biomed. Res. Found., Washington, D.C.), gap open penalty=10, and gapextension penalty=1. For sequences of less than 35 amino acids inlength, optimal parameters are PAM30 matrix (Dayhoff, M. O., supra), gapopen penalty=9, and gap extension penalty=1.

Once the sequences are optimally aligned, the percent identity of thesubject sequence relative to the query sequence is calculated bycounting the number of positions in the optimal alignment which containidentical residue pairs, divide that by the number of residues in thecomparison length (which, unless otherwise specified, is the number ofresidues in the query sequence), and multiplying the resulting number by100. Referring back to the examples shown in FIG. 7, in each example thesequence designated as the query sequence is 22 amino acids in length.Referring to the alignment of FIG. 7A, 20 pairs of aligned amino acidresidues (shown in boldface) are identical in the optimal alignment ofthe query sequence (upper) with the subject sequence (lower). Thus, thisparticular subject sequence has (20/22)×100=91.1% identity to the querysequence; in other words, the subject sequence in the alignment of FIG.7A has at least 91% amino acid sequence identity to the query sequence.In the alignment shown in FIG. 7C, 18 pairs of amino acid residues(shown in boldface) in the optimal alignment are identical; thus thisparticular subject sequence has (18/22)×100=81.8% identity to the querysequence; in other words, the subject sequence in the alignment of FIG.7C has at least 81% amino acid sequence identity to the query sequence.

As applied to polypeptides, the term “substantial identity” (or“substantially identical”) typically means that when two amino acidsequences (i.e. a query sequence and a subject sequence) are optimallyaligned using the BLASTP algorithm (manually or via computer) usingappropriate parameters described above, the subject sequence has atleast about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more percent amino acid sequence identity to the querysequence. In some instances, the substantial identity exists over acomparison length of at least about 100 amino acid residues, such as, atleast about 110, 120, 125, 130, 135, 140, 145, 150, 155, 156, 157, 158,159, 160, 161, 162, 163, 164, 165, or 166 amino acid residues.

Similarly, as applied in the context of two nucleic acid sequences, theterm substantial identity (or substantially identical) means that whentwo nucleic acid sequences (i.e. a query and a subject sequence) areoptimally aligned using the BLASTN algorithm (manually or via computer)using appropriate parameters described below, the subject sequence hasat least about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more percent nucleic acid sequence identity tothe query sequence. Parameters used for nucleic acid sequence alignmentsare: match reward 1, mismatch penalty −3, gap existence penalty 5, gapextension penalty 2 (substitution matrices are not used in the BLASTNalgorithm). In some instances, the substantial identity exists over acomparison length of at least about 300 nucleotide residues, such as atleast about 330, 360, 375, 390, 405, 420, 435, 450, 465, 480, 483, 486,489, 492, 495, or 498 nucleotides.

Additional Aspects

Any polypeptide of the invention may be present as part of a largerpolypeptide sequence, e.g. a fusion protein, such as occurs upon theaddition of one or more domains or subsequences for stabilization ordetection or purification of the polypeptide. A polypeptide purificationsubsequence may include, e.g., an epitope tag, a FLAG tag, apolyhistidine sequence, a GST fusion, or any otherdetection/purification subsequence or “tag” known in the art. Theseadditional domains or subsequences either have little or no effect onthe activity of the polypeptide of the invention, or can be removed bypost synthesis processing steps such as by treatment with a protease,inclusion of an intein, or the like.

Any polypeptide of the invention may also comprise one or more modifiedamino acid. The modified amino acid may be, e.g., a glycosylated aminoacid, a PEGylated amino acid, a farnesylated amino acid, an acetylatedamino acid, a biotinylated amino acid, an amino acid conjugated to alipid moiety, or an amino acid conjugated to an organic derivatizingagent. The presence of modified amino acids may be advantageous in, forexample, (a) increasing polypeptide serum half-life and/or functional invivo half-life, (b) reducing polypeptide antigenicity, (c) increasingpolypeptide storage stability, or (d) increasing bioavailability, e.g.increasing the AUC_(sc). Amino acid(s) are modified, for example,co-translationally or post-translationally during recombinant production(e.g., N-linked glycosylation at N-X-S/T motifs during expression inmammalian cells) or modified by synthetic means. This aspect isdescribed in more detail in the section herein entitled“INTERFERON-ALPHA CONJUGATES”.

The invention also provides a composition comprising at least onepolypeptide of the invention, and an excipient or carrier. In oneaspect, the composition comprises an isolated or recombinant polypeptidecomprising an amino acid sequence which differs in 0-16 amino acidpositions (such as in 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, or 16 amino acid positions), from one of SEQ ID NOs:1-15 and SEQ IDNOs:44-104 (such as, for example, one of SEQ ID NO:1, SEQ ID NO:3, SEQID NO:8, SEQ ID NO:12, SEQ ID NO:47, or SEQ ID NO:53), plus a carrier orexcipient. The composition may be a composition comprising apharmaceutically acceptable excipient or carrier. Exemplary compositionsand excipients and carriers are described below.

Making Polypeptides

Recombinant methods for producing and isolating polypeptides of theinvention are described herein. In addition to recombinant production,the polypeptides may be produced by direct peptide synthesis usingsolid-phase techniques (see, e.g., Stewart et al. (1969) Solid-PhasePeptide Synthesis, WH Freeman Co, San Francisco; Merrifield J. (1963) JAm Chem Soc 85:2149-2154). Peptide synthesis may be performed usingmanual techniques or by automation. Automated synthesis may be achieved,for example, using Applied Biosystems 431A Peptide Synthesizer (PerkinElmer, Foster City, Calif.) in accordance with the instructions providedby the manufacturer. For example, subsequences may be chemicallysynthesized separately and combined using chemical methods to providefull-length polypeptides or fragments thereof. Alternatively, suchsequences may be ordered from any number of companies which specializein production of polypeptides. Most commonly, polypeptides of theinvention may be produced by expressing coding nucleic acids andrecovering polypeptides, e.g., as described below.

Methods for producing the polypeptides of the invention are alsoincluded. One such method comprises introducing into a population ofcells any nucleic acid of the invention described herein, which isoperatively linked to a regulatory sequence effective to produce theencoded polypeptide, culturing the cells in a culture medium to expressthe polypeptide, and isolating the polypeptide from the cells or fromthe culture medium. An amount of nucleic acid sufficient to facilitateuptake by the cells (transfection) and/or expression of the polypeptideis utilized. The nucleic acid is introduced into such cells by anydelivery method described herein, including, e.g., injection, gene gun,passive uptake, etc. The nucleic acid may be part of a vector, such as arecombinant expression vector, including a DNA plasmid vector, or anyvector described herein. The nucleic acid or vector comprising a nucleicacid of the invention may be prepared and formulated as describedherein, above. Such a nucleic acid or expression vector may beintroduced into a population of cells of a mammal in vivo, or selectedcells of the mammal (e.g., tumor cells) may be removed from the mammaland the nucleic acid expression vector introduced ex vivo into thepopulation of such cells in an amount sufficient such that uptake andexpression of the encoded polypeptide results. Or, a nucleic acid orvector comprising a nucleic acid of the invention is produced usingcultured cells in vitro. In one aspect, the method of producing apolypeptide of the invention comprises introducing into a population ofcells a recombinant expression vector comprising any nucleic acid of theinvention described herein in an amount and formula such that uptake ofthe vector and expression of the encoded polypeptide will result;administering the expression vector into a mammal by anyintroduction/delivery format described herein; and isolating thepolypeptide from the mammal or from a byproduct of the mammal.

Antibodies

In another aspect of the invention, a polypeptide of the invention (oran antigenic fragment thereof) is used to produce antibodies which have,e.g., diagnostic, therapeutic, or prophylactic uses, e.g., related tothe activity, distribution, and expression of polypeptides and fragmentsthereof. Antibodies to polypeptides of the invention may be generated bymethods well known in the art. Such antibodies may include, but are notlimited to, polyclonal, monoclonal, chimeric, humanized, single chain,Fab fragments and fragments produced by a Fab expression library.Antibodies, e.g., those that block receptor binding, are especiallypreferred for therapeutic and/or prophylactic use.

Polypeptides for antibody induction do not require biological activity;however, the polypeptides or peptides should be antigenic. Peptides usedto induce specific antibodies may have an amino acid sequence consistingof at least about 10 amino acids, preferably at least about 15 or 20amino acids or at least about 25 or 30 amino acids. Short stretches of apolypeptide may be fused with another protein, such as keyhole limpethemocyanin, and antibody produced against the chimeric molecule.

Methods of producing polyclonal and monoclonal antibodies are known tothose of skill in the art, and many antibodies are available. See, e.g.,Current Protocols in Immunology, John Colligan et al., eds., Vols. I-IV(John Wiley & Sons, Inc., NY, 1991 and 2001 Supplement); and Harlow andLane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press,NY; Stites et al. (eds.) Basic and Clinical Immunology (4th ed.) LangeMedical Publications, Los Altos, Calif., and references cited therein;and Goding (1986) Monoclonal Antibodies: Principles and Practice (2ded.) Academic Press, New York, N.Y.; and Kohler and Milstein (1975)Nature 256:495-497. Other suitable techniques for antibody preparationinclude selection of libraries of recombinant antibodies in phage orsimilar vectors. See, Huse et al. (1989) Science 246:1275-1281; and Wardet al. (1989) Nature 341:544-546. Specific monoclonal and polyclonalantibodies and antisera will usually bind with a K_(D) of at least about0.1 μM, preferably at least about 0.01 mM or better, and most typicallyand preferably, 0.001 μM or better.

Detailed methods for preparation of chimeric (humanized) antibodies canbe found in U.S. Pat. No. 5,482,856. Additional details on humanizationand other antibody production and engineering techniques can be found inBorrebaeck (ed.) (1995) Antibody Engineering, 2^(nd) Edition Freeman andCompany, NY (Borrebaeck); McCafferty et al. (1996) Antibody Engineering,A Practical Approach IRL at Oxford Press, Oxford, England (McCafferty),and Paul (1995) Antibody Engineering Protocols Humana Press, Towata,N.J. (Paul).

In one aspect, this invention provides for fully humanized antibodiesagainst the polypeptides of the invention or fragments thereof.Humanized antibodies are especially desirable in applications where theantibodies are used as therapeutics and/or prophylactics in vivo inhuman patients. Human antibodies consist of characteristically humanimmunoglobulin sequences. The human antibodies of this invention can beproduced in using a wide variety of methods (see, e.g., Larrick et al.,U.S. Pat. No. 5,001,065, and Borrebaeck McCafferty and Paul, supra, fora review). In one aspect, the human antibodies of the present inventionare produced initially in trioma cells. Genes encoding the antibodiesare then cloned and expressed in other cells, such as nonhuman mammaliancells. The general approach for producing human antibodies by triomatechnology is described by Ostberg et al. (1983), Hybridoma 2:361-367,Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No.4,634,666. The antibody-producing cell lines obtained by this method arecalled triomas because they are descended from three cells—two human andone mouse. Triomas have been found to produce antibody more stably thanordinary hybridomas made from human cells.

Other uses contemplated for polypeptides of the invention are providedthroughout the specification.

Interferon-Alpha Conjugates

In another aspect, the invention relates to a conjugate comprising apolypeptide exhibiting an interferon-alpha activity which comprises anamino acid sequence of any one of SEQ ID NOs: 1-15 and SEQ IDNOs:44-104, and at least one non-polypeptide moiety attached to thepolypeptide, such as e.g., 1-6, 1-5, 1-4, 1-3, e.g. 1 or 2non-polypeptide moieties attached to the polypeptide. It will beunderstood that the conjugate also exhibits an interferon-alpha activity(such as, antiviral activity, T_(H)1 differentiation activity, and/orantiproliferative activity).

In another aspect, the invention relates to a conjugate comprising apolypeptide exhibiting an interferon-alpha activity, which polypeptidecomprises an amino acid sequence that differs from the amino acidsequence of any one of SEQ ID NOs:1-15 and SEQ ID NOs:44-104 (such as,one of SEQ ID NOs:1-15, 47, or 53), in at least one amino acid residueselected from an introduced or removed amino acid residue comprising anattachment group for a non-polypeptide moiety. Examples of amino acidresidues to be introduced and/or removed according to this aspect aredescribed in further detail in the following sections. It will beunderstood that the conjugate itself also exhibits an interferon-alphaactivity.

In another aspect the conjugate comprises an amino acid sequence whichdiffers from the amino acid sequence of any of SEQ ID NOs:1-15 and SEQID NOs:44-104 (such as, one of SEQ ID NOs:1-15, 47, or 53) in 0-16 aminoacid positions (such as in 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, or 16 amino acid positions), e.g. in 0-14 amino acid positions,in 0-12 amino acid positions, in 0-10 amino acid positions, in 0-8 aminoacid positions, in 0-6 amino acid positions, in 0-5 amino acidpositions, in 0-4 amino acid positions, in 0-3 amino acid positions, in0-2 amino acid positions, or in 0-1 amino acid positions. In one aspectof the invention, the amino acid residue comprising an attachment groupfor the non-polypeptide moiety is introduced (e.g., by substitution ofan amino acid residue for a different residue which comprises anattachment group for the non-polypeptide moiety, or by insertion of anadditional amino acid residue which comprises an attachment group forthe non-polypeptide moiety).

The term “conjugate” (or interchangeably “polypeptide conjugate” or“conjugated polypeptide”) is intended to indicate a heterogeneous (inthe sense of composite) molecule formed by the covalent attachment ofone or more polypeptides of the invention to one or more non-polypeptidemoieties. The term “covalent attachment” means that the polypeptide andthe non-polypeptide moiety are either directly covalently joined to oneanother, or else are indirectly covalently joined to one another throughan intervening moiety or moieties, such as a bridge, spacer, or linkagemoiety or moieties. Preferably, a conjugated polypeptide is soluble atrelevant concentrations and conditions, i.e. soluble in physiologicalfluids such as blood. Examples of conjugated polypeptides of theinvention include glycosylated and/or PEGylated polypeptides. The term“non-conjugated polypeptide” may be used to refer to the polypeptidepart of the conjugated polypeptide.

The term “non-polypeptide moiety” is intended to mean a molecule that iscapable of conjugating to an attachment group of the polypeptide.Preferred examples of non-polypeptide moieties include polymermolecules, sugar moieties, lipophilic compounds, or organic derivatizingagents, in particular polymer molecules or sugar moieties. It will beunderstood that the non-polypeptide moiety is linked to the polypeptidethrough an attachment group of the polypeptide. Except where the numberof non-polypeptide moieties, such as polymer molecule(s), attached tothe polypeptide is expressly indicated, every reference to “anon-polypeptide moiety” attached to the polypeptide or otherwise used inthe present invention shall be a reference to one or morenon-polypeptide moieties attached to the polypeptide.

The term “polymer molecule” is defined as a molecule formed by covalentlinkage of two or more monomers, wherein none of the monomers is anamino acid residue. The term “polymer” may be used interchangeably withthe term “polymer molecule”.

The term “sugar moiety” is intended to indicate a carbohydrate moleculeattached by in vivo or in vitro glycosylation, such as N- orO-glycosylation.

An “N-glycosylation site” has the sequence N-X-S/T/C, wherein X is anyamino acid residue except proline, N is asparagine and S/T/C is eitherserine, threonine or cysteine, preferably serine or threonine, and mostpreferably threonine.

An “O-glycosylation site” comprises the OH— group of a serine orthreonine residue.

The term “attachment group” is intended to indicate an amino acidresidue group capable of coupling to the relevant non-polypeptide moietysuch as a polymer molecule or a sugar moiety. Non-limiting examples ofuseful attachment groups and some corresponding non-polypeptide moietiesare provided in Table 4 below.

TABLE 4 Useful attachment groups and examples of correspondingnon-polypeptide moieties Conjugation Attachment Examples of non-method/activated group Amino acid polypeptide moiety PEG Reference —NH₂N-terminus, Lys Polymer, e.g. PEG mPEG-SPA Nektar Inc. 2003 mPEG2-NHSCatalog mPEG2- butryALD —COOH C-terminus, Asp, Polymer, e.g. PEG mPEG-HzNektar Inc. 2003 Glu Sugar moiety In vitro coupling Catalog —SH CysPolymer, e.g. PEG, mPEG-VS Nektar Inc. 2003 Sugar moiety mPEG2-MALCatalog; In vitro coupling Delgado et al, Critical Reviews inTherapeutic Drug Carrier Systems 9(3,4): 249-304 (1992) —OH Ser, Thr,OH—, Lys Sugar moiety In vivo O-linked glycosylation —CONH₂ Asn as partof an Sugar moiety In vivo N-glycosylation glycosylation site AromaticPhe, Tyr, Trp Sugar moiety In vitro coupling residue —CONH₂ Gln Sugarmoiety In vitro coupling Yan and Wold, Biochemistry, 1984, Jul 31;23(16): 3759-65 Aldehyde Oxidized Polymer, e.g. PEG, PEGylation Andreszet al., Ketone carbohydrate PEG-hydrazide 1978, Makromol. Chem. 179:301; WO 92/16555, WO 00/23114 Guanidino Arg Sugar moiety In vitrocoupling Lundblad and Noyes, Chemical Reagents for Protein Modification,CRC Press Inc. Boca Raton, Fl Imidazole ring His Sugar moiety In vitrocoupling As for guanidine

For in vivo N-glycosylation, the term “attachment group” is used in anunconventional way to indicate the amino acid residues constituting anN-glycosylation site (with the sequence N-X-S/T/C, wherein X is anyamino acid residue except proline, N is asparagine and S/T/C is eitherserine, threonine or cysteine, preferably serine or threonine, and mostpreferably threonine). Although the asparagine residue of theN-glycosylation site is the one to which the sugar moiety is attachedduring glycosylation, such attachment cannot be achieved unless theother amino acid residues of the N-glycosylation site is present.Accordingly, when the non-polypeptide moiety is a sugar moiety and theconjugation is to be achieved by N-glycosylation, the term “amino acidresidue comprising an attachment group for the non-polypeptide moiety”as used in connection with alterations of the amino acid sequence of thepolypeptide of the invention is to be understood as one, two or all ofthe amino acid residues constituting an N-glycosylation site is/are tobe altered in such a manner that either a functional N-glycosylationsite is introduced into the amino acid sequence, removed from saidsequence or a functional N-glycosylation site is retained in the aminoacid sequence (e.g. by substituting a serine residue, which alreadyconstitutes part of an N-glycosylation site, with a threonine residueand vice versa).

The term “introduce” (i.e., an “introduced” amino acid residue,“introduction” of an amino acid residue) is primarily intended to meansubstitution of an existing amino acid residue for another amino acidresidue, but may also mean insertion of an additional amino acidresidue.

The term “remove” (i.e., a “removed” amino acid residue, “removal” of anamino acid residue) is primarily intended to mean substitution of theamino acid residue to be removed for another amino acid residue, but mayalso mean deletion (without substitution) of the amino acid residue tobe removed.

The term “amino acid residue comprising an attachment group for thenon-polypeptide moiety” is intended to indicate that the amino acidresidue is one to which the non-polypeptide moiety binds (in the case ofan introduced amino acid residue) or would have bound (in the case of aremoved amino acid residue).

The term “functional in vivo half-life” is used in its normal meaning,i.e. the time at which 50% of the biological activity of the polypeptideis still present in the body/target organ, or the time at which theactivity of the polypeptide is 50% of the initial value. The functionalin vivo half-life may be determined in an experimental animal, such asrat, mice, rabbit, dog or monkey. Preferably, the functional in vivohalf half-life is determined in a non-human primate, such as a monkey.Furthermore, the functional in vivo half-life may be determined for asample that has been administered intravenously or subcutaneously.

As an alternative to determining functional in vivo half-life, “serumhalf-life” may be determined, i.e. the time at which 50% of thepolypeptide circulates in the plasma or bloodstream prior to beingcleared. Determination of serum half-life is often more simple thandetermining the functional in vivo half-life and the magnitude of serumhalf-life is usually a good indication of the magnitude of functional invivo half-life. Alternatively terms to serum half-life include “plasmahalf-life”, “circulating half-life”, “serum clearance”, “plasmaclearance” and “clearance half-life”. The serum half-life may bedetermined as described above in connection with determination offunctional in vivo half-life.

The term “serum” is used in its normal meaning, i.e. as blood plasmawithout fibrinogen and other clotting factors.

The term “increased” as used about the functional in vivo half-life orserum half-life is used to indicate that the relevant half-life of theconjugate of the invention is statistically significantly increasedrelative to that of a reference molecule, such as a wild-typeinterferon-alpha, e.g., a human interferon-alpha, such as one of SEQ IDNO:31-SEQ ID NO:42 (or other huIFN-alpha sequences as described hereinand/or in Allen G. and Diaz M. O. (1996), supra), or the correspondingnon-conjugated polypeptide. Thus, interesting conjugates of theinvention include those which have an increased functional in vivohalf-life or an increased serum half-life as compared to a referencemolecule mentioned above.

The term “AUC_(sc)” or “Area Under the Curve when administeredsubcutaneously” is used in its normal meaning, i.e. as the area underthe interferon-alpha-activity-in-serum vs. time curve, where theconjugated molecule has been administered subcutaneously to anexperimental animal. Once the experimental interferon-alpha activitytime points have been determined, the AUC_(sc) may conveniently becalculated by a computer program, such as GraphPad Prism 3.01.

The term “increased” as used about the AUC_(sc) is used to indicate thatthe Area Under the Curve for a conjugate of the invention, whenadministered subcutaneously, is statistically significantly increasedrelative to that of a reference molecule, such as wild-typeinterferon-alpha, e.g., a human interferon-alpha, such as one of SEQ IDNO:31-SEQ ID NO:42 (or other huIFN-alpha sequences as described hereinand/or in Allen G. and Diaz M. O. (1996), supra), or the correspondingnon-conjugated polypeptide, when determined under comparable conditions.Evidently, the same amount of interferon-alpha activity should beadministered for the conjugate of the invention and the referencemolecule. Consequently, in order to make direct comparisons betweendifferent interferon-alpha molecules, the AUC_(sc) values shouldtypically be normalized, i.e. be expressed as AUC_(sc)/doseadministered.

The term “T_(max,sc)” is used about the time point in theinterferon-alpha-activity-in-serum vs. time curve where the highestinterferon-alpha activity in serum is observed.

It will be understood that while the examples and modifications to theparent polypeptide are generally provided herein in regards to thesequence SEQ ID NO:1, the disclosed modifications may also be made inequivalent amino acid positions of any of the other polypeptides of theinvention (including SEQ ID NOs:2-15 and 44-104 and variants thereof)described above.

By removing and/or introducing amino acid residues comprising anattachment group for the non-polypeptide moiety it is possible tospecifically adapt the polypeptide so as to make the molecule moresusceptible to conjugation to the non-polypeptide moiety of choice, tooptimize the conjugation pattern (e.g. to ensure an optimal distributionof non-polypeptide moieties on the surface of the interferon-alphamolecule and thereby, e.g., effectively shield epitopes and othersurface parts of the polypeptide without significantly impairing thefunction thereof). For instance, by introduction of attachment groups,the interferon-alpha polypeptide is altered in the content of thespecific amino acid residues to which the relevant non-polypeptidemoiety binds, whereby a more efficient, specific and/or extensiveconjugation is achieved. By removal of one or more attachment groups itis possible to avoid conjugation to the non-polypeptide moiety in partsof the polypeptide in which such conjugation is disadvantageous, e.g. toan amino acid residue located at or near a functional site of thepolypeptide (since conjugation at such a site may result in inactivationor reduced interferon-alpha activity of the resulting conjugate due toimpaired receptor recognition). Further, it may be advantageous toremove an attachment group located close to another attachment group.

It will be understood that the amino acid residue comprising anattachment group for a non-polypeptide moiety, whether it be removed orintroduced, is selected on the basis of the nature of thenon-polypeptide moiety and, in some instances, on the basis of theconjugation method to be used. For instance, when the non-polypeptidemoiety is a polymer molecule, such as a polyethylene glycol orpolyalkylene oxide derived molecule, amino acid residues capable offunctioning as an attachment group may be selected from the groupconsisting of cysteine, lysine (and/or the N-terminal amino group of thepolypeptide), aspartic acid, glutamic acid, histidine and arginine. Whenthe non-polypeptide moiety is a sugar moiety, the attachment group is anin vivo or in vitro N- or O-glycosylation site, preferably anN-glycosylation site.

In some instances, when an attachment group for a non-polypeptide moietyis to be introduced into or removed from the interferon-alphapolypeptide, the position of the interferon-alpha polypeptide to bemodified may be conveniently selected as follows:

The position to be modified may be located at the surface of theinterferon-alpha polypeptide, such as a position occupied by an aminoacid residue which has more than 25% of its side chain exposed to thesolvent, such as more than 50% of its side chain exposed to the solvent.Such positions have been identified on the basis of an analysis of a 3Dstructure of the human interferon-alpha 2a molecule as described in the“Materials and Methods” section herein.

Alternatively or additionally, the position to be modified may beidentified on the basis of an analysis of an interferon-alpha proteinsequence family (such as shown in the alignments depicted in FIGS. 2 and4). For the purposes of the following example, SEQ ID NO: 1 as shown inthe top line of the alignment of FIG. 2 may be considered the parentinterferon-alpha to be modified, and the human interferon-alphasequences in the rest of the alignment are considered the other membersof the family. For example, the position to be modified in the parentsequence may be one which, in one or more members of the family otherthan the parent interferon-alpha, is (a) occupied by an amino acidresidue comprising the relevant attachment group (when such amino acidresidue is to be introduced into the parent sequence) or (b) which inthe parent interferon-alpha, but not in one or more other members of thefamily, is occupied by an amino acid residue comprising the relevantattachment group (when such amino acid residue is to be removed from theparent sequence).

In order to determine an optimal distribution of attachment groups, thedistance between amino acid residues located at the surface of theinterferon-alpha molecule was calculated on the basis of a 3D structureof an interferon-alpha polypeptide. More specifically, the distancebetween the CB's of the amino acid residues comprising such attachmentgroups, or the distance between the functional group (NZ for lysine, CGfor aspartic acid, CD for glutamic acid, SG for cysteine) of one and theCB of another amino acid residue comprising an attachment group weredetermined. In case of glycine, CA was used instead of CB. In theinterferon-α polypeptide part of a conjugate of the invention, any ofsaid distances may be more than 8 Å, such as more than 10 Å in order toavoid or reduce heterogeneous conjugation and to provide a uniformdistribution of attachment groups, e.g. with the aim of epitopeshielding.

Furthermore, in the interferon-alpha polypeptide part of a conjugate ofthe invention, in some instances attachment groups located at or nearthe receptor binding sites of interferon-alpha are removed, such as bysubstitution of the amino acid residue comprising such group. In someinstances, amino acid residues comprising an attachment group for anon-polypeptide moiety, such as cysteine or lysine, are often notintroduced at or near the receptor binding site of the interferon alphamolecule.

Another approach for modifying an interferon-alpha polypeptide is toshield and thereby modify or destroy or otherwise inactivate an epitopepresent in the parent interferon-alpha, by conjugation to anon-polypeptide moiety. Epitopes of interferon-alpha polypeptides may beidentified by use of methods known in the art, also known as epitopemapping, see e.g. Romagnoli et al., J. Biol. Chem., 1999, 380(5):553-9,DeLisser H M, Methods Mol Biol, 1999, 96:11-20, Van de Water et al.,Clin Immunol Immunopathol, 1997, 85(3):229-35, Saint-Remy J M,Toxicology, 1997, 119(1):77-81, and Lane D P and Stephen C W, Curr OpinImmunol, 1993, 5(2):268-71. One method is to establish a phage displaylibrary expressing random oligopeptides of, e.g., 9 amino acid residues.IgG1 antibodies from specific antisera towards human interferon-alphaare purified by immunoprecipitation and the reactive phages areidentified by immunoblotting. By sequencing the DNA of the purifiedreactive phages, the sequence of the oligopeptide can be determinedfollowed by localization of the sequence on the 3D-structure of theinterferon-alpha. Alternatively, epitopes can be identified according tothe method described in U.S. Pat. No. 5,041,376. The thereby identifiedregion on the structure constitutes an epitope that then can be selectedas a target region for introduction of an attachment group for thenon-polypeptide moiety. Preferably, at least one epitope, such as two,three or four epitopes of interferon-alpha are shielded by anon-polypeptide moiety according to the present invention. Accordingly,in one aspect, the conjugate of the invention has at least one shieldedepitope as compared to a wild type human interferon-alpha, including anycommercially available interferon-alpha. This may be done byintroduction of an attachment group for a non-polypeptide moiety into aposition located in the vicinity of (i.e. within 4 amino acid residuesin the primary sequence or within about 10 Å in the tertiary sequence)of a given epitope. The 10 Å distance is measured between CB's (CA's incase of glycine). Such specific introductions are described in thefollowing sections.

In case of removal of an attachment group, the relevant amino acidresidue comprising such group and occupying a position as defined abovemay be substituted with a different amino acid residue that does notcomprise an attachment group for the non-polypeptide moiety in question,or may be deleted. Removal of an N-glycosylation group, may also beaccomplished by insertion or removal of an amino acid reside within themotif N-X-S/T/C.

In case of introduction of an attachment group, an amino acid residuecomprising such group is introduced into the position, such as bysubstitution of the amino acid residue occupying such position.

The exact number of attachment groups available for conjugation andpresent in the interferon-alpha polypeptide is dependent on the effectdesired to be achieved by conjugation. The effect to be obtained is,e.g., dependent on the nature and degree of conjugation (e.g. theidentity of the non-polypeptide moiety, the number of non-polypeptidemoieties desirable or possible to conjugate to the polypeptide, wherethey should be conjugated or where conjugation should be avoided, etc.).For instance, if reduced immunogenicity is desired, the number (andlocation of) attachment groups should be sufficient to shield most orall epitopes. This is normally obtained when a greater proportion of theinterferon-alpha polypeptide is shielded. Effective shielding ofepitopes is normally achieved when the total number of attachment groupsavailable for conjugation is in the range of 1-6 attachment groups,e.g., 1-5, such as in the range of 1-3, such as 1, 2, or 3 attachmentgroups.

Functional in vivo half-life is i.a. dependent on the molecular weightof the conjugate, and the number of attachment groups needed forproviding increased half-life thus depends on the molecular weight ofthe non-polypeptide moiety in question. Some such conjugates comprise1-6, e.g., 1-5, such as 1-3, e.g. 1, 2, or 3 non-polypeptide moietieseach having a MW of about 2-40 kDa, such as about 2 kDa, about 5 kDa,about 12 kDa, about 15 kDa, about 20 kDa, about 30 kDa, or about 40 kDa.

In the conjugate of the invention, some, most, or substantially allconjugatable attachment groups are occupied by the relevantnon-polypeptide moiety.

The conjugate of the invention may exhibit one or more of the followingimproved properties:

For example, the conjugate may exhibit a reduced immunogenicity ascompared to a human interferon-alpha (such as any of the polypeptidesdefined herein as SEQ ID NO:31-42, SEQ ID NO:32+R23K, or any otherhuIFN-alpha described herein and/or in Allen G. and Diaz M. O. (1996),supra) or as compared to the corresponding non-conjugated polypeptide,e.g. a reduction of at least 10%, such as a reduction of at least of25%, such as a reduction of at least of 50%, e.g. a reduction of atleast 75% compared to the non-conjugated polypeptide or compared to ahuman interferon-alpha.

In another aspect the conjugate may exhibit a reduced reaction or noreaction with neutralizing antibodies from patients treated with a humaninterferon-alpha (such as any of the polypeptides defined herein as SEQID NO:31-42, SEQ ID NO:32+R23K, or any other huIFN-alpha describedherein and/or in Allen G. and Diaz M. O. (1996), supra) or as comparedto the corresponding non-conjugated polypeptide, e.g. a reduction ofneutralisation of at least 10%, such as at least of 25%, such as of atleast 50%, e.g., at least 75%.

In another aspect of the invention the conjugate may exhibit anincreased functional in vivo half-life and/or increased serum half-lifeas compared to a reference molecule such as a human interferon-alpha(e.g. any of the polypeptides defined herein as SEQ ID NO:31-42, SEQ IDNO:32+R23K, or any other huIFN-alpha described herein and/or in Allen G.and Diaz M. O. (1996), supra) or as compared to the correspondingnon-conjugated polypeptide. Particular preferred conjugates are suchconjugates where the ratio between the functional in vivo half-life (orserum half-life) of said conjugate and the functional in vivo half-life(or serum half-life) of said reference molecule is at least 1.25, suchas at least 1.50, such as at least 1.75, such as at least 2, such as atleast 3, such as at least 4, such as at least 5, such as at least 6,such as at least 7, such as at least 8. As mentioned above, thehalf-life is conveniently determined in an experimental animal, such asrat or monkey, and may be based on intravenously or subcutaneouslyadministration.

In a further aspect the conjugate may exhibit an increasedbioavailability as compared to a reference molecule such as a humaninterferon-alpha (e.g. any of the polypeptides defined herein as SEQ IDNO:31-42, SEQ ID NO:32+R23K, or any other huIFN-alpha described hereinand/or in Allen G. and Diaz M. O. (1996), supra) or the correspondingnon-conjugated polypeptide. For example, the conjugate may exhibit anincreased AUC_(sc) as compared to a reference molecule such as a humaninterferon-alpha or the corresponding non-conjugated polypeptide. Thus,exemplary conjugates are such conjugates where the ratio between theAUC_(sc) of said conjugate and the AUC_(sc) of said reference moleculeis at least 1.25, such as at least 1.5, such as at least 2, such as atleast 3, such as at least 4, such as at least 5 or at least 6, such asat least 7, such as at least 8, such as at least 9 or at least 10, suchas at least 12, such as at least 14, e.g. at least 16, at least 18 or atleast 20 when administered subcutaneously, in particular whenadministered subcutaneously in an experimental animal such as rat ormonkey. Analogously, some conjugates of the invention are suchconjugates wherein the ratio between T_(max) for said conjugate andT_(max) for said reference molecule, such as a human interferon-alpha orthe corresponding non-conjugated polypeptide, is at least 1.2, such asat least 1.4, e.g. at least 1.6, such as at least 1.8, such as at least2, e.g. at least 2.5, such as at least 3, such as at least 4, e.g. atleast 5, such as at least 6, such as at least 7, e.g. at least 8, suchas at least 9, such as at least 10, when administered subcutaneously, inparticular when administered subcutaneously in an experimental animalsuch as rat or monkey.

In some instances, the magnitude of the antiviral activity of aconjugate of the invention may be reduced (e.g. by at least about 75%,at least about 50%, at least about 25%, at least about 10%) or increased(e.g. by at least about 10%) or is about equal (e.g. within about +/−10%or about +/−5%) to that of a human interferon-alpha (e.g. any of thepolypeptides identified herein as SEQ ID NO:31-42, SEQ ID NO:32+R23K, orany other huIFN-alpha described herein and/or in Allen G. and Diaz M. O.(1996), supra) or to that of the corresponding non-conjugatedpolypeptide. In some instances the degree of antiviral activity ascompared to antiproliferative activity of a conjugate of the inventionmay vary, and thus be higher, lower or about equal to that of a humaninterferon-alpha or to that of the corresponding non-conjugatedpolypeptide.

Conjugate of the Invention where the Non-Polypeptide Moiety Binds to aCysteine Residue

In another aspect, the invention relates to a conjugate exhibiting aninterferon-alpha activity and comprising at least one non-polypeptidemoiety conjugated to at least one cysteine residue of aninterferon-alpha, the amino acid sequence of which differs in 0-16 aminoacid positions from that of a parent interferon-alpha polypeptide, suchas an interferon-alpha polypeptide comprising the amino acid sequence ofany of SEQ ID NOs:1-15 and 44-104 (such as, one of SEQ ID NO:1, SEQ IDNO:3, SEQ ID NO:8, SEQ ID NO: 12, SEQ ID NO:47 or SEQ ID NO:53), in thatat least one cysteine residue has been introduced, such as bysubstitution or insertion, into a position that is occupied in theparent interferon-alpha by an amino acid residue that is exposed to thesurface of the molecule, preferably one that has at least 25%, such asat least 50% of its side chain exposed to the surface. Typically, theconjugate comprises an amino acid sequence which differs from the aminoacid sequence of any one of, e.g., SEQ ID NOs:1-15, 47 or 53, in 1-16amino acid positions (such as in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, or 16 amino acid positions), e.g. in 1-14 amino acidpositions, in 1-12 amino acid positions, in 1-10 amino acid positions,in 1-8 amino acid positions, in 1-6 amino acid positions, in 1-5 aminoacid positions, in 1-4 amino acid positions, in 1-3 amino acid positionsor in 1-2 amino acid positions.

Some conjugates of the invention comprise a polypeptide sequencecomprising one or more of the following substitutions, relative to SEQID NO:1, which introduces a cysteine residue into a position which ispredicted to be exposed at the surface of the molecule with more than a25% fractional ASA: D2C, L3C, P4C, Q5C, T6C, H7C, S8C, L9C, G10C, R12C,R13C, M16C, A19C, Q20C, R22C, R23C, 124C, S25C, L26C, F27C, S28C, L30C,K31C, R33C, H34C, D35C, R37C, Q40C, E41C, E42C, D44C, N46C, H47C, Q49C,K50C, V51C, Q52C, E59C, Q62C, Q63C, N66C, S69C, T70C, K71C, N72C, S74C,A75C, D78C, E79C, T80C, L81C, E83C, K84C, 187C, F90C, Q91C, N94C, D95C,E97C, A98C, V100C, M101C, Q102C, E103C, V104C, G105C, E107C, E108C,T109C, P110C, L111C, M112C, N113C, V114C, D115C, L118C, R121C, K122C,Q125C, R126C, T128C, L129C, T132C, K133C, K134C, K135C, Y136C, S137C,P138C, A146C, M149C, R150C, S153C, F154C, N157C, Q159C, K160C, R161C,L162C, R163C, R164C, K165C and E166C, said amino acid residue positionsrelative to SEQ ID NO:1. In some instances, among the above-mentionedpositions, one or more of the amino acid residues at positions 47, 51and 133 are not substituted with cysteine.

For example, some such conjugates of the invention comprise apolypeptide sequence comprising one or more of the followingsubstitutions, relative to SEQ ID NO:1, which introduces a cysteineresidue into a position which is predicted to be exposed at the surfaceof the molecule with more than a 50% fractional ASA: D2C, L3C, P4C, Q5C,T6C, H7C, S8C, L9C, R12C, R13C, M16C, A19C, S25C, F27C, S28C, K31C,R33C, H34C, D35C, R37C, E41C, D44C, N46C, H47C, Q49C, K50C, N66C, K71C,A75C, D78C, E79C, T80C, E83C, K84C, 187C, F90C, Q91C, N94C, D95C, M101C,Q102C, E103C, G105C, E107C, E108C, T109C, P110C, L111C, V114C, D115C,L118C, R121C, K122C, Q125C, R126C, L129C, T132C, K133C, K135C, P138C,R150C, K160C, L162C, R163C, R164C, K165C and E166C, said amino acidresidue positions relative to SEQ ID NO:1. In some instances, one orboth of the amino acid residues at positions 47 and 133 are not are notsubstituted with cysteine.

As indicated above, in some instances it may be preferable to introducecysteine residues outside of potential receptor binding sites ofinterferon-alpha, i.e., outside of about positions 29-40, 79-96, and124-141, position numbering relative to SEQ ID NO:1. Thus, in someinstances the one or more cysteine substitutions are selected from thegroup consisting of D2C, L3C, P4C, Q5C, T6C, H7C, S8C, L9C, G10C, R12C,R13C, M16C, A19C, Q20C, R22C, R23C, 124C, S25C, L26C, F27C, S28C, E41C,E42C, D44C, N46C, H47C, Q49C, K50C, V51C, Q52C, E59C, Q62C, Q63C, N66C,S69C, T70C, K71C, N72C, S74C, A75C, E97C, A98C, V100C, M101C, Q102C,E103C, V104C, G105C, E107C, E108C, T109C, P110C, L111C, M112C, N113C,V114C, D115C, L118C, R121C, K122C, A146C, M149C, R150C, S153C, F154C,N157C, Q159C, K160C, R161C, L162C, R163C, R164C, K165C and E166C(positions which are predicted to be exposed at the surface of themolecule with more than a 25% fractional ASA and are not part of theputative receptor binding sites), relative to SEQ ID NO:1. In someinstances, one or both of positions 47 and 51 are not substituted withcysteine.

In other aspects the cysteine substitution is selected from the groupconsisting of: D2C, L3C, P4C, Q5C, T6C, H7C, S8C, L9C, R12C, R13C, M16C,A19C, S25C, F27C, S28C, E41C, D44C, N46C, H47C, Q49C, K50C, N66C, K71C,A75C, M101C, Q102C, E103C, G105C, E107C, E108C, T109C, P110C, L111C,V114C, D115C, L118C, R121C, K122C, R150C, K160C, L162C, R163C, R164C,K165C and E166C (positions which are predicted to be exposed at thesurface of the molecule with more than a 50% fractional ASA and are notpart of the putative receptor binding sites), relative to SEQ ID NO:1.In some instances, position 47 is not substituted with a cysteine.

Some such conjugates of the invention comprise a polypeptide sequencecontaining one or more of the substitutions S25C, S28C, L30C, K31C,N46C, K71C, S74C, A75C, E79C, E107C, E108C, T132C, K133C, P138C, andK135C, relative to SEQ ID NO:1.

In another aspect, the one or more cysteine residue is introduced at ornear the C-terminus either by substitution (for example, Q159C, K160C,R161C, L162C, R163C, R164C, K165C or E166C, relative to SEQ ID NO:1) orby insertion (for example, E166EC, also referred to herein as 167C). Itwill be understood that cysteine residues may also be introduced, eitherby substitution or by insertion, in C-terminally truncated fragments ofthe interferon-alpha molecules described herein.

In some instances, only a single cysteine residue is introduced in orderto avoid formation of disulfide bridges between two or more introducedcysteine residues.

In interferon alphas, disulfide bonds are formed between cysteines atpositions 1/99 and 29/139. The disulfide bond 29/139 is essential forbiological activity, while the 1/99 bond can be reduced withoutsignificantly affecting biological activity (Beilharz M. W. et al.(1986) J. Interferon Res. 6(6):677-685). Thus, in another aspect of theinvention one of C1 or C99 is removed, preferably by substitution, e.g.C1S or C99S, thereby leaving the other cysteine residue available forconjugation to a non-polypeptide moiety.

Non-polypeptide moieties contemplated in this aspect of the inventioninclude polymer molecules, such as any of the molecules mentioned in thesection entitled “Conjugation to a polymer molecule”, such as PEG ormPEG. The conjugation between the cysteine-containing polypeptide andthe polymer molecule may be achieved in any suitable manner, e.g. asdescribed in the section entitled “Conjugation to a polymer molecule”,e.g. in using a one step method or in the stepwise manner referred to insaid section. An exemplary method for PEGylating the interferon-alphapolypeptide is to covalently attach PEG to cysteine residues usingcysteine-reactive PEGs. A number of highly specific, cysteine-reactivePEGs with different groups (e.g. orthopyridyl-disulfide (OPSS),maleimide (MAL) and vinylsulfone (VS)) and different size linear orbranched PEGs (e.g., 2-40 kDa, such as 2 kDa, 5 kDa, 12 kDa, 15 kDa, 20kDa, 30 kDa, or 40 kDa) are commercially available, e.g. from NektarTherapeutics Inc., Huntsville, Ala., USA, or SunBio, Anyang City, SouthKorea.

It is to be understood that while the examples of modifications to theparent polypeptide are generally provided herein relative to thesequence SEQ ID NO:1 (or relative to some other specified sequence), thedisclosed modifications may also be made in equivalent amino acidpositions of any of the other polypeptides of the invention (includingSEQ ID NOs:2-15 and SEQ ID NOs:44-104 and variants thereof) describedherein. Thus, as an example, the substitution H47C relative to SEQ IDNO:1 is understood to correspond to Q47C in SEQ ID NO:5, and so on.

Conjugate of the Invention where the Non-Polypeptide Moiety Binds to aLysine Residue

In another aspect, the invention relates to a conjugate exhibiting aninterferon-alpha activity and comprising at least one non-polypeptidemoiety conjugated to at least one lysine residue, and/or to theN-terminal amino group, of an interferon-alpha polypeptide comprising asequence selected from SEQ ID NOs:1-15 and 44-104, or comprising asequence which differs from any of SEQ ID NOs: 1-15 and 44-104 (such as,one of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ IDNO:47 or SEQ ID NO:53), in 0-16 amino acid positions (such as in 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acidpositions), e.g. in 0-14 amino acid positions, in 0-12 amino acidpositions, in 0-10 amino acid positions, in 0-8 amino acid positions, in0-6 amino acid positions, in 0-5 amino acid positions, in 0-4 amino acidpositions, in 0-3 amino acid positions, in 0-2 amino acid positions orin 0-1 amino acid position. Some conjugates according to this aspectcomprise at least one removed lysine residue and/or at least one removedhistidine residue, and/or at least one introduced lysine residue.

Some conjugates of the invention comprise a polypeptide sequencecomprising a substitution of an amino acid residue for a different aminoacid residue, or a deletion of an amino acid residue, which removes oneor more lysines, e.g., K31, K50, K71, K84, K122, K133, K134, K135, K160,and/or K165 (relative to SEQ ID NO:1) from any polypeptide of theinvention such as, for example, one of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:8, SEQ ID NO:12, SEQ ID NO:47 or SEQ ID NO:53. The one or more lysineresidue(s) to be removed may be substituted with any other amino acid,may be substituted with an Arg (R) or Gln (O), or may be deleted. Somesuch conjugates comprise the substitutions K31R+K122R; K31R+K133R;K122R+K133R; or K31R+K122R+K133R. Other exemplary substitutions includeK71E; K84E; K133E/G; and K160E.

In instances where amine-reactive conjugation chemistries are employed,it may be advantageous to avoid or to minimize the potential forconjugation to histidine residues. Therefore, some conjugates of theinvention comprise a polypeptide sequence comprising a substitution or adeletion which removes one or more histidines, e.g., H7, H11, H34,and/or H47 (relative to SEQ ID NO:1) from any polypeptide sequence ofthe invention such as, for example, one of SEQ ID NO:1, SEQ ID NO:3, SEQID NO:8, SEQ ID NO: 12, SEQ ID NO:47 or SEQ ID NO:53. The one or morehistidine residue(s) to be removed may be substituted with any otheramino acid, may be substituted with an Arg (R) or Gln (O), or may bedeleted. Some such conjugates comprise the substitutions H34Q; H47Q; orH34Q+H47Q.

Alternatively, or in addition, some conjugates of the invention comprisea polypeptide sequence comprising a modification which introduces alysine into a position that is occupied in the parent sequence (e.g.,one of SEQ ID NOs:1-15, 47, or 53) by an amino acid residue that isexposed to the surface of the molecule, e.g., one that has at least 25%,such as at least 50% of its side chain exposed to the surface. Some suchconjugates comprise a polypeptide sequence comprising one or more of thefollowing substitutions, relative to SEQ ID NO:1, which introduces alysine residue into a position which is predicted to be exposed at thesurface of the molecule with more than a 25% fractional ASA: D2K, L3K,P4K, Q5K, T6K, H7K, S8K, L9K, G10K, R12K, R13K, M16K, A19K, Q20K, R22K,R23K, I24K, S25K, L26K, F27K, S28K, L30K, R33K, H34K, D35K, R37K, Q40K,E41K, E42K, D44K, N46K, H47K, Q49K, V51K, Q52K, E59K, Q62K, Q63K, N66K,S69K, T70K, N72K, S74K, A75K, D78K, E79K, T80K, L81K, E83K, 187K, F90K,Q91K, N94K, D95K, E97K, A98K, V100K, M101K, Q102K, E103K, V104K, G105K,E107K, E108K, T109K, P110K, L111K, M112K, N113K, V114K, D115K, L118K,R121K, Q125K, R126K, T128K, L129K, T132K, Y136K, S137K, P138K, A146K,M149K, R150K, S153K, F154K, N157K, Q159K, R161K, L162K, R163K, R164K,and E166K, said amino acid residue positions relative to SEQ ID NO: 1.In some instances, among the above-mentioned positions, one or more ofthe amino acid residues at positions 47, 51, 52, and 154 are notsubstituted with lysine.

Some such conjugates of the invention comprise a polypeptide sequencecomprising one or more of the following substitutions, relative to SEQID NO:1, which introduces a lysine residue into a position which ispredicted to be exposed at the surface of the molecule with more than a50% fractional ASA: D2K, L3K, P4K, Q5K, T6K, H7K, S8K, L9K, R12K, R13K,M16K, A19K, S25K, F27K, S28K, R33K, H34K, D35K, R37K, E41K, D44K, N46K,H47K, Q49K, N66K, A75K, D78K, E79K, T80K, E83K, 187K, F90K, Q91K, N94K,D95K, M101K, Q102K, E103K, G105K, E107K, E108K, T109K, P110K, L111K,V114K, D115K, L118K, R121K, Q125K, R126K, L129K, T132K, P138K, R150K,L162K, R163K, R164K and E166K, said amino acid residue positionsrelative to SEQ ID NO:1. In some instances, among the above-mentionedpositions, positions 47 is not substituted with lysine.

As indicated above, in some instances it may be preferable to introducelysine residues outside of potential receptor binding sites ofinterferon-alpha, i.e., outside of about positions 29-40, 79-96, and124-141, position numbering relative to SEQ ID NO:1. Thus, in someinstances the one or more lysine substitutions are selected from thegroup consisting of D2K, L3K, P4K, Q5K, T6K, H7K, S8K, L9K, G10K, R12K,R13K, M16K, A19K, Q20K, R22K, R23K, I24K, S25K, L26K, F27K, S28K, E41K,E42K, D44K, N46K, H47K, Q49K, V51K, Q52K, E59K, Q62K, Q63K, N66K, S69K,T70K, N72K, S74K, A75K, E97K, A98K, V100K, M101K, Q102K, E103K, V104K,G105K, E107K, E108K, T109K, P110K, L111K, M112K, N113K, V114K, D115K,L118K, R121K, A146K, M149K, R150K, S153K, F154K, N157K, Q159K, R161K,L162K, R163K, R164K, and E166K (residues having more than 25% of theside chain exposed to the surface and not forming part of the putativereceptor binding sites), relative to SEQ ID NO:1. In some instances, oneor more of positions 47, 51, and 154 are not substituted with lysine.

In some instances the one or more lysine substitutions are selected fromthe group consisting of: D2K, L3K, P4K, Q5K, T6K, H7K, S8K, L9K, R12K,R13K, M16K, A19K, S25K, F27K, S28K, E41K, D44K, N46K, H47K, Q49K, N66K,A75K, M101K, Q102K, E103K, G105K, E107K, E108K, T109K, P110K, L111K,V114K, D115K, L118K, R121K, R150K, L162K, R163K, R164K and E166K(residues having more than 50% of the side chain exposed to the surfacean not forming part of the putative receptor binding sites), relative toSEQ ID NO:1. In some instances, position 47 is not substituted with alysine.

Non-polypeptide moieties contemplated for this aspect of the inventioninclude polymer molecules, such as any of the molecules mentioned in thesection entitled “Conjugation to a polymer molecule”, such as PEG ormPEG. The conjugation between the lysine-containing polypeptide and thepolymer molecule may be achieved in any suitable manner, e.g. asdescribed in the section entitled “Conjugation to a polymer molecule”,e.g. in using a one step method or in the stepwise manner referred to insaid section. An exemplary method for PEGylating the interferon-alphapolypeptide is to covalently attach PEG to lysine residues usinglysine-reactive PEGs. A number of highly specific, lysine-reactive PEGs(such as for example, succinimidyl propionate (SPA), succinimidylbutanoate (SBA), N-hydroxylsuccinimide (NHS), and aldehyde (e.g.,ButyrALD)) and different size linear or branched PEGs (e.g., 2-40 kDa,such as 2 kDa, 5 kDa, 12 kDa, 15 kDa, 20 kDa, 30 kDa, or 40 kDa) arecommercially available, e.g. from Nektar Therapeutics Inc., Huntsville,Ala., USA, or SunBio, Anyang City, South Korea.

It is to be understood that while the examples of modifications to theparent polypeptide are generally provided herein relative to thesequence SEQ ID NO:1 (or relative to some other specified sequence), thedisclosed modifications may also be made in equivalent amino acidpositions of any of the other polypeptides of the invention (includingSEQ ID NOs:2-15 and SEQ ID NOs:44-104 and variants thereof) describedherein. Thus, as an example, the substitution H47K relative to SEQ IDNO:1 is understood to correspond to Q47K in SEQ ID NO:5, and so on.

Conjugate of the Invention where the Non-Polypeptide Moiety is a SugarMoiety

In another aspect, the invention relates to a conjugate exhibitinginterferon-alpha activity and comprising at least one sugar moietyconjugated to an interferon-alpha polypeptide, the amino acid sequenceof which differs from that of a parent interferon-alpha polypeptide,such as any one of SEQ ID NOs:1-15 and SEQ ID NOs:44-104 (such as one ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47 or SEQID NO:53), 1-16 amino acid positions, in that at least one glycosylationsite, preferably an in vivo N-glycosylation site, has been introduced,preferably by substitution, into a position that in the parentinterferon-alpha polypeptide is occupied by an amino acid residue thatis exposed to the surface of the molecule, e.g. one that has at least25%, such as at least 50% of its side chain exposed to the surface.Typically, the conjugate comprises an amino acid sequence which differsfrom the amino acid sequence of any of, for example, SEQ ID NOs:1-15,47, or 53, in 1-16 amino acid positions (such as in 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid positions), e.g. in 1-14amino acid positions, in 1-12 amino acid positions, in 1-10 amino acidpositions, in 1-8 amino acid positions, in 1-6 amino acid positions, in1-5 amino acid positions, in 1-4 amino acid positions, in 1-3 amino acidpositions or in 1-2 amino acid positions.

The N-glycosylation site is introduced in such a way that the N-residue(Asn) of said site is located in the designated position. Analogously,an O-glycosylation site is introduced so that the S (Ser) or T (Thr)residue making up such site is located in said position. It should beunderstood that when the term “at least 25% (or 50%) of its side chainexposed to the surface” is used in connection with introduction of an invivo N-glycosylation site this term refers to the surface accessibilityof the amino acid side chain in the position where the sugar moiety isactually attached. In many cases it will be necessary to introduce aserine or a threonine residue in position +2 relative to the asparagineresidue to which the sugar moiety is actually attached and thesepositions, where the serine or threonine residues are introduced, areallowed to be buried, i.e. to have less than 25% (or 50%) of their sidechains exposed to the surface of the molecule.

Some conjugates of the invention comprise a polypeptide sequencecomprising one or more of the following substitutions, relative to SEQID NO:1, which introduces an N-glycosylation site into a position whichis predicted to be exposed at the surface of the molecule with more thana 25% fractional ASA: D2N+P4S/T, L3N+Q5S/T, P4Q, P4Q+T6S, Q5N+H7S/T,T6N, T6N+S8T, H7N+L9S/T, S8N+G10S/T, L9N+H11S/T, G10N+R12S/T, R12N,R12N+T14S, R13N+M15S/T, M16N+L18S/T, A19N+M21S/T, Q20N+R22S/T,R22N+124S/T, R23N, R23N+S25T, 124N+L26S/T, S25N+F27S/T, L26N, L26N+S28T,S28N+L30S/T, L30N+D32S/T, K31N+R33S/T, R33N+D35S/T, H34N+F36S/T,D35N+R37S/T, R37N+P39S/T, Q40N+E42S/T, E41N+F43S/T, E42N+D44S/T,D44N+N46S/T, F48S/T, H47N+Q49S/T, Q49N+V51S/T, K50N+Q52S/T, V51N+A53S/T,Q52N+154S/T, E59N+M61S/T, Q62N, Q62N+T64S, Q63N+F65S/T, F68S/T,S69N+K71S/T, T70N+N72S/T, K71N, K71N+S73T, S74T, S74N+A76S/T,A75N+W77S/T, D78N, D78N+T80S, E79N+L81S/T, T80N+L82S/T, L81N+E83S/T,E83N+F85S/T, K84N+Y86S/T, 187N+L89S/T, F90N+Q92S/T, Q91N+M93S/T, L96S/T,D95N+E97S/T, E97N+C99S/T, A98N+V100S/T, V100N+Q102S/T, M101N+E103S/T,Q102N+V104S/T, E103N+G105S/T, V104N+V106S/T, G105N+E107S/T, E107,E107N+T109S, E108N+P110S/T, L111N+N113S/T, M112N+V114S/T, N113N+D115S/T,V114N, V114N+S116T, D115N+1117S/T, L118N+V120S/T, R121N+Y123S/T,K122N+F124S/T, Q125N+1127S/T, R126N, R126N+T128S, T128N+Y130S/T,L129N+L131S/T, T132N+K134S/T, K133N+K135S/T, K134N+Y136S/T, K135N,K135N+S137T, Y136N+P138S/T, P138N, P138N+S140T, A146N+1148S/T, M149N,M149N+S151T, R150N+F152S/T, S153N, S153N+S155T, F154N+F156S/T, Q159S/T,K160N+L162S/T, R161N+R163S/T, L162N+R164S/T, R163N+K165S/T andR164N+E166S/T, relative to SEQ ID NO:1. In some instances, among theabove-mentioned positions, the amino acid residues at one or more ofpositions 47, 51, 133 and 140 are not modified as shown above. S/Tindicates a substitution to a serine or threonine residue, preferably athreonine residue.

Some conjugates of the invention comprise a polypeptide sequencecomprising one or more of the following substitutions, relative to SEQID NO:1, which introduces an N-glycosylation site into a position whichis predicted to be exposed at the surface of the molecule with more thana 50% fractional ASA: D2N+P4S/T, L3N+Q5S/T, P4Q, P4Q+T6S, Q5N+H7S/T,T6N, T6N+S8T, H7N+L9S/T, S8N+G10S/T, L9N+H11S/T, R12N, R12N+T14S,R13N+M15S/T, M16N+L18S/T, A19N+M21S/T, S25N+F27S/T, S28N+L30S/T,R33N+D35S/T, H34N+F36S/T, D35N+R37S/T, R37N+P39S/T, E41N+F43S/T,D44N+N46S/T, F48S/T, H47N+Q49S/T, Q49N+V51S/T, K50N+Q52S/T, F68S/T,K71N, K71N+S73T, A75N+W77S/T, D78N, D78N+T80S, E79N+L81S/T, T80N+L82S/T,E83N+F85S/T, K84N+Y86S/T, 187N+L89S/T, F90N+Q92S/T, Q91N+M93S/T, L96S/T,D95N+E97S/T, M101N+E103S/T, Q102N+V104S/T, E103N+G105S/T, G105N+E107S/T,E107, E107N+T109S, E108N+P110S/T, L111N+N113S/T, V114N, V114N+S116T,D115N+117S/T, L118N+V120S/T, R121N+Y123S/T, K122N+F124S/T,Q125N+1127S/T, R126N, R126N+T128S, L129N+L131S/T, T132N+K134S/T,K133N+K135S/T, K135N, K135N+S137T, P138N, P138N+S140T, R150N+F152S/T,K160N+L162S/T, L162N+R164S/T, R163N+K165S/T and R164N+E166S/T, relativeto SEQ ID NO:1. In some instances, among the above-mentioned positions,the amino acid residues at one or more of positions 47, 51, 133 and 140are not modified as described above. S/T indicates a substitution to aserine or threonine residue, preferably a threonine residue.

In some instances it may be preferable to introduce N-glycosylationsite(s) outside of potential receptor binding sites of interferon-alpha,i.e., outside of about positions 29-40, 79-96, and 124-141, positionnumbering relative to SEQ ID NO:1. Thus, the substitution(s) leading tointroduction of one or more N-glycosylation site may include one or moreof D2N+P4S/T, L3N+Q5S/T, P4Q, P4Q+T6S, Q5N+H7S/T, T6N, T6N+S8T,H7N+L9S/T, S8N+G10S/T, L9N+H11S/T, G10N+R12S/T, R12N, R12N+T14S,R13N+M15S/T, M16N+L18S/T, A19N+M21S/T, Q20N+R22S/T, R22N+124S/T, R23N,R23N+S25T, 124N+L26S/T, S25N+F27S/T, L26N, L26N+S28T, S28N+L30S/T,E41N+F43S/T, E42N+D44S/T, D44N+N46S/T, F48S/T, H47N+Q49S/T, Q49N+V51S/T,K50N+Q52S/T, V51N+A53S/T, Q52N+154S/T, E59N+M61S/T, Q62N, Q62N+T64S,Q63N+F65S/T, F68S/T, S69N+K71S/T, T70N+N72S/T, K71N, K71N+S73T, S74T,S74N+A76S/T, A75N+W77S/T, E97N+C99S/T, A98N+V100S/T, V100N+Q102S/T,M101N+E103S/T, Q102N+V104S/T, E103N+G105S/T, V104N+V106S/T,G105N+E107S/T, E107, E107N+T109S, E108N+P110S/T, L111N+N113S/T,M112N+V114S/T, N113N+D115S/T, V114N, V114N+S1±6T, D115N+1117S/T,L118N+V120S/T, R121N+Y123S/T, K122N+F124S/T, A146N+1148S/T, M149N,M149N+S151T, R150N+F152S/T, S153N, S153N+S155T, F154N+F156S/T, Q159S/T,K160N+L162S/T, R161N+R163S/T, L162N+R164S/T, R163N+K165S/T andR164N+E166S/T (residues having more than 25% of the side chain exposedto the surface an not forming part of the putative binding sites). Insome instances, among the above-mentioned positions, the amino acidresidues at one or both of positions 47 and 51 are not modified as shownabove. S/T indicates a substitution to a serine or threonine residue,preferably a threonine residue.

In some instances the substitution(s) are selected from the groupconsisting of: D2N+P4S/T, L3N+Q5S/T, P4Q, P4Q+T6S, Q5N+H7S/T, T6N,T6N+S8T, H7N+L9S/T, S8N+G10S/T, L9N+H11S/T, R12N, R12N+T14S,R13N+M15S/T, M16N+L18S/T, A19N+M21S/T, S25N+F27S/T, S28N+L30S/T,E41N+F43S/T, D44N+N46S/T, F48S/T, H47N+Q49S/T, Q49N+V51S/T, K50N+Q52S/T,F68S/T, K71N, K71N+S73T, A75N+W77S/T, M101N+E103S/T, Q102N+V104S/T,E103N+G105S/T, G105N+E107S/T, E107, E107N+T109S, E108N+P110S/T,L111N+N113S/T, V114N, V114N+S116T, D115N+1117S/T, L118N+V120S/T,R121N+Y123S/T, K122N+F124S/T, R150N+F152S/T, K160N+L162S/T,L162N+R164S/T, R163N+K165S/T and R164N+E166S/T (residues having morethan 50% of the side chain exposed to the surface an not forming part ofthe putative binding sites). In some instances, among theabove-mentioned positions, the amino acid residues at one or both ofpositions 47 and 51 are not modified as shown above. S/T indicates asubstitution to a serine or threonine residue, preferably a threonineresidue.

In order to obtain efficient utilization of the introducedN-glycosylation site it is desirable to select any of theabove-mentioned substitutions within about the 125 N-terminal amino acidresidues, such as within about the 100 N-terminal amino acid residues,e.g. within the 75 N-terminal amino acid residues or within the 50N-terminal amino acid residues.

When the interferon-alpha polypeptide part of a conjugate of theinvention is glycosylated, it may contain a single introduced in vivoglycosylation site, such as a single introduced in vivo N-glycosylationsite. However, in order to obtain efficient shielding of epitopespresent on the surface of the parent polypeptide it may be desirablethat the polypeptide comprises more than one in vivo glycosylation site,such as 2-5 in vivo glycosylation sites, e.g. 2, 3, 4, or 5 in vivoglycosylation sites.

It is to be understood that while the examples of modifications to theparent polypeptide are generally provided herein relative to thesequence SEQ ID NO:1 (or relative to some other specified sequence), thedisclosed modifications may also be made in equivalent amino acidpositions of any of the other polypeptides of the invention (includingSEQ ID NOs:2-15 and SEQ ID NOs:44-104 and variants thereof) describedherein. Thus, as an example, the substitution H47N+Q49S/T relative toSEQ ID NO:1 is understood to correspond to Q47N+Q49S/T in SEQ ID NO:5,and so on.

Non-Polypeptide Moiety of the Conjugate of the Invention

As indicated above, the non-polypeptide moiety of the conjugate of theinvention is generally selected from the group consisting of a polymermolecule, a lipophilic compound, a sugar moiety (e.g., by way of in vivoN-glycosylation) and an organic derivatizing agent. All of these agentsmay confer desirable properties to the polypeptide part of theconjugate, such as reduced immunogenicity, increased functional in vivohalf-life, increased serum half-life, increased bioavailability and/orincreased AUC_(sc). The polypeptide part of the conjugate is oftenconjugated to only one type of non-polypeptide moiety, but may also beconjugated to two or more different types of non-polypeptide moieties,e.g. to a polymer molecule and a sugar moiety, etc. The conjugation totwo or more different non-polypeptide moieties may be donesimultaneously or sequentially. The choice of non-polypeptidemoiety/moieties, depends especially on the effect desired to be achievedby the conjugation. For instance, sugar moieties have been foundparticularly useful for reducing immunogenicity, whereas polymermolecules such as PEG are of particular use for increasing functional invivo half-life and/or serum half-life. Using a combination of a polymermolecule and a sugar moiety may enhance the reduction in immunogenicityand the increase in functional in vivo or serum half-life.

In the following sections “Conjugation to a lipophilic compound”,“Conjugation to a polymer molecule”, “Conjugation to a sugar moiety” and“Conjugation to an organic derivatizing agent” conjugation to specifictypes of non-polypeptide moieties is described.

Conjugation to a Lipophilic Compound

For conjugation to a lipophilic compound the following polypeptidegroups may function as attachment groups: the N-terminus or C-terminusof the polypeptide, the hydroxy groups of the amino acid residues Ser,Thr or Tyr, the ε-amino group of Lys, the SH group of Cys or thecarboxyl group of Asp and Glu. The polypeptide and the lipophiliccompound may be conjugated to each other either directly or by use of alinker. The lipophilic compound may be a natural compound such as asaturated or unsaturated fatty acid, a fatty acid diketone, a terpene, aprostaglandin, a vitamin, a carotenoid or steroid, or a syntheticcompound such as a carbon acid, an alcohol, an amine and sulphonic acidwith one or more alkyl, aryl, alkenyl or other multiple unsaturatedcompounds. The conjugation between the polypeptide and the lipophiliccompound, optionally through a linker may be done according to methodsknown in the art, e.g. as described by Bodanszky in Peptide Synthesis,John Wiley, New York, 1976 and in WO 96/12505.

Conjugation to a Polymer Molecule

The polymer molecule to be coupled to the polypeptide may be anysuitable polymer molecule, such as a natural or synthetic homo-polymeror heteropolymer, typically with a molecular weight in the range ofabout 300-100,000 Da, such as about 1000-50,000 Da, e.g. in the range ofabout 1000-40,000 Da. More particularly, the polymer molecule, such asPEG, in particular mPEG, will typically have a molecular weight of about2, 5, 10, 12, 15, 20, 30, 40 or 50 kDa, in particular a molecular weightof about 5 kDa, about 10 kDa, about 12 kDa, about 15 kDa, about 20 kDa,about 30 kDa or about 40 kDa. The PEG molecule may be branched (e.g.,mPEG2), or may be unbranched (i.e., linear).

When used about polymer molecules herein, the word “about” indicates anapproximate average molecular weight and reflects the fact that therewill normally be a certain molecular weight distribution in a givenpolymer preparation.

Examples of homo-polymers include a polyol (i.e. poly-OH), a polyamine(i.e. poly-NH₂) and a polycarboxylic acid (i.e. poly-COOH). Ahetero-polymer is a polymer which comprises one or more differentcoupling groups, such as a hydroxyl group and an amine group.

Examples of suitable polymer molecules include polymer moleculesselected from the group consisting of polyalkylene oxide (PAO),including polyalkylene glycol (PAG), such as polyethylene glycol (PEG)and polypropylene glycol (PPG), branched PEGs (PEG2), poly-vinyl alcohol(PVA), poly-carboxylate, poly-(vinylpyrolidone), polyethylene-co-maleicacid anhydride, polystyrene-co-malic acid anhydride, dextran includingcarboxymethyl-dextran, or any other biopolymer suitable for reducingimmunogenicity and/or increasing functional in vivo half-life and/orserum half-life. Generally, polyalkylene glycol-derived polymers arebiocompatible, non-toxic, non-antigenic, non-immunogenic, have variouswater solubility properties, and are easily excreted from livingorganisms.

PEG is the preferred polymer molecule to be used, since it has only fewreactive groups capable of cross-linking compared to e.g.polysaccharides such as dextran. In particular, monofunctional PEG, e.g.monomethoxypolyethylene glycol (mPEG), is of interest since its couplingchemistry is relatively simple (only one reactive group is available forconjugating with attachment groups on the polypeptide). Consequently,the risk of cross-linking is eliminated, the resulting polypeptideconjugates are more homogeneous and the reaction of the polymermolecules with the polypeptide is easier to control.

To effect covalent attachment of the polymer molecule(s) to thepolypeptide, the hydroxyl end groups of the polymer molecule must beprovided in activated form, i.e. with reactive functional groups(examples of which include primary amino groups, hydrazide (HZ), thiol,succinate (SUC), succinimidyl succinate (SS), succinimidyl succinamide(SSA), succinimidyl propionate (SPA), succinimidyl butanoate (SBA),succinimidyl carboxymethylate (SCM), benzotriazole carbonate (BTC),N-hydroxysuccinimide (NHS), aldehyde, nitrophenylcarbonate (NPC), andtresylate (TRES)). Suitably activated polymer molecules are commerciallyavailable, e.g. from Nektar Therapeutics, Inc., Huntsville, Ala., USA;PolyMASC Pharmaceuticals plc, UK; or SunBio Corporation, Anyang City,South Korea. Alternatively, the polymer molecules can be activated byconventional methods known in the art, e.g. as disclosed in WO 90/13540.Specific examples of activated linear or branched polymer moleculessuitable for use in the present invention are described in the NektarTherapeutics, Inc. 2003 Catalog (“Nektar Molecule Engineering:Polyethylene Glycol and Derivatives for Advanced Pegylation, Catalog2003”), incorporated by reference herein. Specific examples of activatedPEG polymers include the following linear PEGs: NHS-PEG, SPA-PEG,SSPA-PEG, SBA-PEG, SS-PEG, SSA-PEG, SC-PEG, SG-PEG, SCM-PEG, NOR-PEG,BTC-PEG, EPOX-PEG, NCO-PEG, NPC-PEG, CDI-PEG, ALD-PEG, TRES-PEG, VS-PEG,OPSS-PEG, IODO-PEG, and MAL-PEG, and branched PEGs, such as PEG2-NHS,PEG2-MAL, and those disclosed in U.S. Pat. No. 5,932,462 and U.S. Pat.No. 5,643,575, both of which are incorporated herein by reference.Furthermore, the following publications, incorporated herein byreference, disclose useful polymer molecules and/or PEGylationchemistries: U.S. Pat. No. 5,824,778, U.S. Pat. No. 5,476,653, WO97/32607, EP 229,108, EP 402,378, U.S. Pat. No. 4,902,502, U.S. Pat. No.5,281,698, U.S. Pat. No. 5,122,614, U.S. Pat. No. 5,219,564, WO92/16555, WO 94/04193, WO 94/14758, WO 94/17039, WO 94/18247, WO94/28024, WO 95/00162, WO 95/11924, WO 95/13090, WO 95/33490, WO96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO 99/32134, WO99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO 95/06058, EP 439508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131, U.S. Pat. No.5,736,625, WO 98/05363, EP 809 996, U.S. Pat. No. 5,629,384, WO96/41813, WO 96/07670, U.S. Pat. No. 5,473,034, U.S. Pat. No. 5,516,673,EP 605 963, U.S. Pat. No. 5,382,657, EP 510 356, EP 400 472, EP 183 503and EP 154 316.

The conjugation of the polypeptide and the activated polymer moleculesis conducted by use of any conventional method, e.g. as described in thefollowing references (which also describe suitable methods foractivation of polymer molecules): Harris and Zalipsky, eds.,Poly(ethylene glycol) Chemistry and Biological Applications, AZC,Washington; R. F. Taylor, (1991), “Protein immobilisation. Fundamentaland applications”, Marcel Dekker, N.Y.; S. S. Wong, (1992), “Chemistryof Protein Conjugation and Crosslinking”, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), “Immobilized Affinity Ligand Techniques”,Academic Press, N.Y.

For PEGylation of cysteine residues the polypeptide is usually treatedwith a reducing agent, such as dithiothreitol (DDT) prior to PEGylation.The reducing agent is subsequently removed by any conventional method,such as by desalting. Conjugation of PEG to a cysteine residue typicallytakes place in a suitable buffer at pH 6-9 at temperatures varying from4° C. to 25° C. for periods up to about 16 hours. Examples of activatedPEG polymers for coupling to cysteine residues include the followinglinear and branched PEGs: vinylsulfone-PEG (PEG-VS), such asvinylsulfone-mPEG (mPEG-VS); orthopyridyl-disulfide-PEG (PEG-OPSS), suchas orthopyridyl-disulfide-mPEG (mPEG-OPSS); and maleimide-PEG (PEG-MAL),such as maleimide-mPEG (mPEG-MAL) and branched maleimide-mPEG2(mPEG2-MAL).

Pegylation of lysines often employs PEG-N-hydroxylsuccinimide (e.g.,mPEG-NHS or mPEG2-NHS), or esters such as PEG succinimidyl propionate(e.g., mPEG-SPA) or PEG succinimidyl butanoate (e.g., mPEG-SBA). One ormore PEGs can be attached to a protein within 30 minutes at pH 8-9.5 atroom temperature if about equimolar amounts of PEG and protein aremixed. A molar ratio of PEG to protein amino groups of 1-5 to 1 willusually suffice. Increasing pH increases the rate of reaction, whilelowering pH reduces the rate of reaction. These highly reactive activeesters can couple at physiological pH, but less reactive derivativestypically require higher pH. Low temperatures may also be employed if alabile protein is being used. Under low temperature conditions, a longerreaction time may be used.

N-terminal PEGylation is facilitated by the difference between the pKavalues of the α-amino group of the N-terminal amino acid (˜7.6 to 8.0)and the ε-amino group of lysine (˜10). PEGylation of the N-terminalamino group often employs PEG-aldehydes (such as mPEG-propionaldehyde ormPEG-butylaldehyde), which are more selective for amines and thus areless likely to react with the imidazole group of histidine; in addition,PEG reagents used for lysine conjugation (such as mPEG-SPA or mPEG-SBA)may also be used for conjugation of the N-terminal amine. Conjugation ofa PEG-aldehyde to the N-terminal amino group typically takes place in asuitable buffer (such as, 100 mM sodium acetate or 100 mM sodiumbisphosphate buffer with 20 mM sodium cyanoborohydride) at pH ˜5.0overnight at temperatures varying from about 4° C. to 25° C. UsefulN-terminal PEGylation methods and chemistries are also described in U.S.Pat. No. 5,985,265 and U.S. Pat. No. 6,077,939, both incorporated hereinby reference.

Typically, linear PEG or mPEG polymers will have a molecular weight ofabout 5 kDa, about 10 kDa, about 12 kDa, about 15 kDa, about 20 kDa, orabout 30 kDa. Branched PEG (PEG2 or mPEG2) polymers will typically havea molecular weight of about 10 kDa, about 20 kDa, or about 40 kDa. Insome instances, the higher-molecular weight branched PEG2 reagents, suchas 20 kDa or 40 kDa PEG2, including e.g. mPEG2-NHS for lysinePEGylation, mPEG2-MAL for cysteine PEGylation, or MPEG2-aldehyde forN-terminal PEGylation (all available from Nektar Therapeutics, Inc,Huntsville Ala.), may be used. The branched structure of the PEG2Compound results in a relatively large molecular volume, so fewerattached molecules (or, one attached molecule) may impart the desiredcharacteristics of the PEGylated molecule.

The skilled person will be aware that the activation method and/orconjugation chemistry to be used depends on the attachment group(s) ofthe interferon-alpha polypeptide as well as the functional groups of thepolymer (e.g., being amino, hydroxyl, carboxyl, aldehyde or sulfhydryl).The PEGylation may be directed towards conjugation to all availableattachment groups on the polypeptide (i.e. such attachment groups thatare exposed at the surface of the polypeptide) or may be directedtowards specific attachment groups, e.g. cysteine residues, lysineresidues, or the N-terminal amino group. Furthermore, the conjugationmay be achieved in one step or in a stepwise manner (e.g. as describedin WO 99/55377).

In some instances, the polymer conjugation is performed under conditionsaiming at reacting as many of the available polymer attachment groups aspossible with polymer molecules. This is achieved by means of a suitablemolar excess of the polymer in relation to the polypeptide. Typicalmolar ratios of activated polymer molecules to polypeptide are up toabout 1000-1, such as up to about 200-1 or up to about 100-1. In somecases, the ratio may be somewhat lower, however, such as up to about50-1, 10-1 or 5-1. Also equimolar ratios may be used.

It is also contemplated according to the invention to couple the polymermolecules to the polypeptide through a linker. Suitable linkers are wellknown to the skilled person. A preferred example is cyanuric chloride(Abuchowski et al., (1977), J. Biol. Chem., 252, 3578-3581; U.S. Pat.No. 4,179,337; Shafer et al., (1986), J. Polym. Sci. Polym. Chem. Ed.,24, 375-378).

Subsequent to the conjugation residual activated polymer molecules areblocked according to methods known in the art, e.g. by addition ofprimary amine to the reaction mixture, and the resulting inactivatedpolymer molecules removed by a suitable method.

Covalent in vitro coupling of a sugar moiety to amino acid residues ofinterferon-alpha may be used to modify or increase the number or profileof sugar substituents. Depending on the coupling mode used, thecarbohydrate(s) may be attached to a) arginine and histidine (Lundbladand Noyes, Chemical Reagents for Protein Modification, CRC Press Inc.Boca Raton, Fla.), b) free carboxyl groups (e.g. of the C-terminal aminoacid residue, asparagine or glutamine), c) free sulfhydryl groups suchas that of cysteine, d) free hydroxyl groups such as those of serine,threonine, tyrosine or hydroxyproline, e) aromatic residues such asthose of phenylalanine or tryptophan or f) the amide group of glutamine.These amino acid residues constitute examples of attachment groups for asugar moiety, which may be introduced and/or removed in theinterferon-alpha polypeptide. Suitable methods of in vitro coupling aredescribed in WO 87/05330 and in Aplin et al., CRC Crit. Rev. Biochem.,pp. 259-306, 1981. The in vitro coupling of sugar moieties or PEG toprotein- and peptide-bound Gln-residues can also be carried out bytransglutaminases (TGases), e.g. as described by Sato et al., 1996Biochemistry 35, 13072-13080 or in EP 725145.

Coupling to a Sugar Moiety

In order to achieve in vivo glycosylation of an interferon-alphapolypeptide that has been modified by introduction of one or moreglycosylation sites (see the section “Conjugates of the inventionwherein the non-polypeptide moiety is a sugar moiety”), the nucleotidesequence encoding the polypeptide part of the conjugate is inserted in aglycosylating, eukaryotic expression host. The expression host cell maybe selected from fungal (filamentous fungal or yeast), insect, mammaliananimal cells, from transgenic plant cells or from transgenic animals.Furthermore, the glycosylation may be achieved in the human body whenusing a nucleotide sequence encoding the polypeptide part of a conjugateof the invention or a polypeptide of the invention in gene therapy. Inone aspect the host cell is a mammalian cell, such as a CHO cell, a COScell, a BHK or HEK cell, e.g. HEK293, or an insect cell, such as an SF9cell, or a yeast cell, e.g. Saccharomyces cerevisiae, Pichia pastoris orany other suitable glycosylating host, e.g. as described further below.Optionally, sugar moieties attached to the interferon-α polypeptide byin vivo glycosylation are further modified by use ofglycosyltransferases, e.g. using the GlycoAdvance™ technology marketedby Neose, Horsham, Pa., USA. Thereby, it is possible to, e.g., increasethe sialyation of the glycosylated interferon-alpha polypeptidefollowing expression and in vivo glycosylation by CHO cells.

Coupling to an Organic Derivatizing Agent

Covalent modification of the interferon-alpha polypeptide may beperformed by reacting (an) attachment group(s) of the polypeptide withan organic derivatizing agent. Suitable derivatizing agents and methodsare well known in the art. For example, cysteinyl residues most commonlyare reacted with α-haloacetates (and corresponding amines), such aschloroacetic acid or chloroacetamide, to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteinyl residues also are derivatizedby reaction with bromotrifluoroacetone, α-bromo-β-(4-imidozoyl)propionicacid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyldisulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide is also useful; the reaction ispreferably performed in 0.1 M sodium cacodylate at pH 6.0. Lysinyl andamino terminal residues are reacted with succinic or other carboxylicacid anhydrides. Derivatization with these agents has the effect ofreversing the charge of the lysinyl residues. Other suitable reagentsfor derivatizing α-amino-containing residues include imidoesters such asmethyl picolinimidate; pyridoxal phosphate; pyridoxal;chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea;2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineguanidino group. Carboxyl side groups (aspartyl or glutamyl orC-terminal amino acid residue) are selectively modified by reaction withcarbodiimides (R—N═C═N—R′), where R and R′ are different alkyl groups,such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Blocking of a Functional Site

Since excessive polymer conjugation may lead to a loss of activity ofthe interferon-α polypeptide to which the polymer is conjugated, it maybe advantageous to remove attachment groups located at the functionalsite or to block the functional site prior to conjugation. These latterstrategies constitute further aspects of the invention (the firststrategy being exemplified further above, e.g. by removal of lysineresidues which may be located close to a functional site). Morespecifically, according to the second strategy the conjugation betweenthe interferon-alpha polypeptide and the non-polypeptide moiety isconducted under conditions where the functional site of the polypeptideis blocked by a helper molecule capable of binding to the functionalsite of the polypeptide. Preferably, the helper molecule is one whichspecifically recognizes a functional site of the polypeptide, such as areceptor, in particular the type I interferon receptor. Alternatively,the helper molecule may be an antibody, in particular a monoclonalantibody recognizing the interferon-alpha polypeptide. In particular,the helper molecule may be a neutralizing monoclonal antibody.

The polypeptide is allowed to interact with the helper molecule beforeeffecting conjugation. This ensures that the functional site of thepolypeptide is shielded or protected and consequently unavailable forderivatization by the non-polypeptide moiety such as a polymer.Following its elution from the helper molecule, the conjugate betweenthe non-polypeptide moiety and the polypeptide can be recovered with atleast a partially preserved functional site.

The subsequent conjugation of the polypeptide having a blockedfunctional site to a polymer, a lipophilic compound, an organicderivatizing agent or any other compound is conducted in the normal way,e.g. as described in the sections above entitled “Conjugation to . . .”.

Irrespective of the nature of the helper molecule to be used to shieldthe functional site of the polypeptide from conjugation, it is desirablethat the helper molecule is free from or comprises only a few attachmentgroups for the non-polypeptide moiety of choice in parts of the moleculewhere the conjugation to such groups would hamper the desorption of theconjugated polypeptide from the helper molecule. Hereby, selectiveconjugation to attachment groups present in non-shielded parts of thepolypeptide can be obtained and it is possible to reuse the helpermolecule for repeated cycles of conjugation. For instance, if thenon-polypeptide moiety is a polymer molecule such as PEG, which has theepsilon amino group of a lysine or N-terminal amino acid residue as anattachment group, it is desirable that the helper molecule issubstantially free from conjugatable epsilon amino groups, preferablyfree from any epsilon amino groups. Accordingly, in some instances thehelper molecule is a protein or peptide capable of binding to thefunctional site of the polypeptide, which protein or peptide is freefrom any conjugatable attachment groups for the non-polypeptide moietyof choice.

In a further aspect the helper molecule is first covalently linked to asolid phase such as column packing materials, for instance Sephadex oragarose beads, or a surface, e.g. reaction vessel. Subsequently, thepolypeptide is loaded onto the column material carrying the helpermolecule and conjugation carried out according to methods known in theart, e.g. as described in the sections above entitled “Conjugation to .. . ”. This procedure allows the polypeptide conjugate to be separatedfrom the helper molecule by elution. The polypeptide conjugate is elutedby conventional techniques under physico-chemical conditions that do notlead to a substantive degradation of the polypeptide conjugate. Thefluid phase containing the polypeptide conjugate is separated from thesolid phase to which the helper molecule remains covalently linked. Theseparation can be achieved in other ways: For instance, the helpermolecule may be derivatized with a second molecule (e.g. biotin) thatcan be recognized by a specific binder (e.g. streptavidin). The specificbinder may be linked to a solid phase thereby allowing the separation ofthe polypeptide conjugate from the helper molecule-second moleculecomplex through passage over a second helper-solid phase column whichwill retain, upon subsequent elution, the helper molecule-secondmolecule complex, but not the polypeptide conjugate. The polypeptideconjugate may be released from the helper molecule in any appropriatefashion. De-protection may be achieved by providing conditions in whichthe helper molecule dissociates from the functional site of theinterferon-α to which it is bound. For instance, a complex between anantibody to which a polymer is conjugated and an anti-idiotypic antibodycan be dissociated by adjusting the pH to an acid or alkaline pH.

Conjugation of a Tagged Interferon-Alpha Polypeptide

In another aspect the interferon-alpha polypeptide is expressed as afusion protein with a tag, i.e. an amino acid sequence or peptide madeup of typically 1-30, such as 1-20 or 1-15 or 1-10 or 1-5 amino acidresidues, e.g. added to the N-terminus or to the C-terminus of thepolypeptide. Besides allowing for fast and easy purification, the tag isa convenient tool for achieving conjugation between the taggedpolypeptide and the non-polypeptide moiety. In particular, the tag maybe used for achieving conjugation in microtiter plates or othercarriers, such as paramagnetic beads, to which the tagged polypeptidecan be immobilised via the tag. The conjugation to the taggedpolypeptide in, e.g., microtiter plates has the advantage that thetagged polypeptide can be immobilised in the microtiter plates directlyfrom the culture broth (in principle without any purification) andsubjected to conjugation. Thereby, the total number of process steps(from expression to conjugation) can be reduced. Furthermore, the tagmay function as a spacer molecule ensuring an improved accessibility tothe immobilised polypeptide to be conjugated. The conjugation using atagged polypeptide may be to any of the non-polypeptide moietiesdisclosed herein, e.g. to a polymer molecule such as PEG.

The identity of the specific tag to be used is not critical as long asthe tag is capable of being expressed with the polypeptide and iscapable of being immobilised on a suitable surface or carrier material.A number of suitable tags are commercially available, e.g. from UnizymeLaboratories, Denmark. Antibodies against such tags are commerciallyavailable, e.g. from ADI, Aves Lab and Research Diagnostics.

Polynucleotides of the Invention

The invention provides isolated or recombinant nucleic acids (alsoreferred to herein as polynucleotides), collectively referred to as“nucleic acids (or polynucleotides) of the invention”, which encodepolypeptides of the invention. The polynucleotides of the invention areuseful in a variety of applications. As discussed above, thepolynucleotides are useful in producing polypeptides of the invention.In addition, polynucleotides of the invention can be incorporated intoexpression vectors useful for gene therapy, DNA vaccination, andimmunotherapy, as described in more detail below.

In one aspect, the invention provides isolated or recombinant nucleicacids that each comprise a polynucleotide sequence selected from: (a) apolynucleotide sequence selected from SEQ ID NOS:16-30, or acomplementary polynucleotide sequence thereof; (b) a polynucleotidesequence which encodes a polypeptide selected from SEQ ID NOS:1-15 and44-104, or a complementary polynucleotide sequence thereof.

The invention also provides isolated or recombinant nucleic acids thateach comprise a polynucleotide sequence which encodes a polypeptidecomprising a sequence which differs in 0-16 amino acid positions (suchas in 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 aminoacid positions), e.g. in 0-16 positions, 0-15 positions, 0-14 positions,0-13 positions, 0-12 positions, 0-11 positions, 0-10 positions, 0-9positions, 0-8 positions, 0-7 positions, 0-6 positions, 0-5 positions,0-4 positions, 0-3 positions, 0-2 positions, or 0-1 positions, from anyone of SEQ ID NOs:1-15 and SEQ ID NOs:44-104 (such as one of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47 or SEQ IDNO:53). In some instances the encoded polypeptide exhibits aninterferon-alpha activity.

The invention also provides isolated or recombinant nucleic acids thateach comprise a polynucleotide sequence which encodes a polypeptidecomprising a sequence having at least about 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% or more amino acid sequence identity to anyone of SEQ ID NOs:1-15 and SEQ ID NOs:44-104 (such as one of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:47 or SEQ IDNO:53). In some instances the encoded polypeptide exhibits aninterferon-alpha activity.

The invention also provides isolated or recombinant nucleic acids thateach comprise a polynucleotide sequence which encodes a polypeptidewhich is a variant of a parent interferon-alpha polypeptide, the encodedvariant comprising a sequence which differs from the parentinterferon-alpha polypeptide sequence in least one amino acid position,wherein the variant sequence comprises one or more of His at position47, Val at position 51, Phe at position 55, Leu at position 56, Tyr atposition 58, Lys at position 133, and at position Ser140, the positionnumbering relative to that of SEQ ID NO:1. In some instances the parentinterferon-alpha polypeptide sequence is a sequence of anaturally-occurring human interferon-alpha (such as any one of SEQ IDNO:31-SEQ ID NO:42, or SEQ ID NO:32+R23K, or other huIFN-alpha sequenceas described herein and/or in Allen G. and Diaz M. O. (1996), supra), oris a sequence of a non-naturally occurring (i.e., synthetic)interferon-alpha, such as IFN-alpha Con1 (SEQ ID NO:43). In someinstances, the variant sequence differs from the parent polypeptidesequence in 1-16 amino acid positions (such as in 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid positions), e.g. in 1-10amino acid positions, in 1-5 amino acid positions, or in 1-3 amino acidpositions. In some instances, the variant exhibits an interferon-alphaactivity.

In another aspect, the invention provides isolated or recombinantnucleic acids that each comprise a polynucleotide sequence whichhybridizes under highly stringent conditions over substantially theentire length of one of SEQ ID NOs:16-30, which polynucleotide sequenceencodes a polypeptide exhibiting an interferon alpha activity.

Additional Aspects

Any of the nucleic acids of the invention (which includes thosedescribed above) may encode a fusion protein comprising at least oneadditional amino acid sequence, such as, for example, asecretion/localization sequence, a sequence useful for solubilization orimmobilization (e.g., for cell surface display) of the polypeptide, asequence useful for detection and/or purification of the polypeptide(e.g., a polypeptide purification subsequence, such as an epitope tag, apolyhistidine sequence, and the like). In another aspect, the inventionprovides cells comprising one or more of the nucleic acids of theinvention. Such cells may express one or more polypeptides encoded bythe nucleic acids of the invention.

The invention also provides vectors comprising any of the nucleic acidsof the invention. Such vectors may comprise a plasmid, a cosmid, aphage, a virus, or a fragment of a virus. Such vectors may comprise anexpression vector, and, if desired, the nucleic acid is operably linkedto a promoter, including those discussed herein and below. Furthermore,in another aspect, the invention provides compositions comprising anexcipient or carrier and at least one of any of the nucleic acids of theinvention, or vectors, cells, or host comprising such nucleic acids.Such composition may be pharmaceutical compositions, and the excipientor carrier may be a pharmaceutically acceptable excipient or carrier.

The invention also includes compositions comprising two or more nucleicacids of the invention, or fragments thereof (e.g., as substrates forrecombination). The composition can comprise a library of recombinantnucleic acids, where the library contains at least 2, at least 3, atleast 5, at least 10, at least 20, at least 50, or at least 100 or morenucleic acids described above. The nucleic acids are optionally clonedinto expression vectors, providing expression libraries.

The nucleic acids of the invention and fragments thereof, as well asvectors comprising such polynucleotides, may be employed for therapeuticor prophylactic uses in combination with a suitable carrier, such as apharmaceutical carrier. Such compositions comprise a therapeuticallyand/or prophylactically effective amount of the compound, and apharmaceutically acceptable carrier or excipient. Such a carrier orexcipient includes, but is not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration. Methods ofadministering nucleic acids, polypeptides, and proteins are well knownin the art, and are further discussed below.

The invention also includes compositions produced by digesting one ormore of any of the nucleic acids of the invention with a restrictionendonuclease, an RNAse, or a DNAse (e.g., as is performed in certain ofthe recombination formats noted above); and compositions produced byfragmenting or shearing one or more nucleic acids of the invention bymechanical means (e.g., sonication, vortexing, and the like), which canalso be used to provide substrates for recombination in the methodsdescribed herein. The invention also provides compositions produced bycleaving at least one of any of the nucleic acids of the invention. Thecleaving may comprise mechanical, chemical, or enzymatic cleavage, andthe enzymatic cleavage may comprise cleavage with a restrictionendonuclease, an RNAse, or a DNAse.

Also included in the invention are compositions produced by a processcomprising incubating one or more of the fragmented nucleic acids of theinvention in the presence of ribonucleotide or deoxyribonucleotidetriphosphates and a nucleic acid polymerase. This resulting compositionforms a recombination mixture for many of the recombination formatsnoted above. The nucleic acid polymerase may be an RNA polymerase, a DNApolymerase, or an RNA-directed DNA polymerase (e.g., a “reversetranscriptase”); the polymerase can be, e.g., a thermostable DNApolymerase (e.g., VENT, TAQ, or the like).

Similarly, compositions comprising sets of oligonucleotidescorresponding to more than one nucleic acids of the invention are usefulas recombination substrates and are a feature of the invention. Forconvenience, these fragmented, sheared, or oligonucleotide synthesizedmixtures are referred to as fragmented nucleic acid sets.

The invention also provides an isolated or recombinant nucleic acidencoding a polypeptide that exhibits an interferon-alpha activity,produced by mutating or recombining at least one nucleic acid of theinvention.

Making Polynucleotides

Polynucleotides, oligonucleotides, and nucleic acid fragments of theinvention can be prepared by standard solid-phase methods, according toknown synthetic methods. Typically, fragments of up to about 100 basesare individually synthesized, then joined (e.g., by enzymatic orchemical ligation methods, or polymerase mediated recombination methods)to form essentially any desired continuous sequence. For example, thepolynucleotides and oligonucleotides of the invention can be prepared bychemical synthesis using, e.g., classical phosphoramidite methoddescribed by, e.g., Beaucage et al. (1981) Tetrahedron Letters22:1859-69, or the method described by Matthes et al. (1984) EMBO J.3:801-05, e.g., as is typically practiced in automated syntheticmethods. According to the phosphoramidite method, oligonucleotides aresynthesized, e.g., in an automatic DNA synthesizer, purified, annealed,ligated and cloned into appropriate vectors.

In addition, essentially any polynucleotide can be custom ordered fromany of a variety of commercial sources, such as Operon Technologies Inc.(Alameda, Calif.) and many others. Similarly, peptides and antibodiescan be custom ordered from any of a variety of sources, e.g., CeltekPeptides (Nashville, Tenn.); Washington Biotechnology, Inc. (BaltimoreMd.); Global Peptide Services (Ft. Collin Colo.), and many others.

Certain polynucleotides of the invention may also be obtained byscreening cDNA libraries (e.g., libraries generated by recombininghomologous nucleic acids as in typical recursive sequence recombinationmethods) using oligonucleotide probes that can hybridize to orPCR-amplify polynucleotides which encode interferon-alpha polypeptidesand fragments of those polypeptides. Procedures for screening andisolating cDNA clones are well-known to those of skill in the art. Suchtechniques are described in, e.g., Berger and Kimmel, Guide to MolecularCloning Techniques, Methods in Enzymol. Vol. 152, Acad. Press, Inc., SanDiego, Calif. (“Berger”); Sambrook, supra, and Current Protocols inMolecular Biology, Ausubel, supra. Some polynucleotides of the inventioncan be obtained by altering a naturally occurring sequence, e.g., bymutagenesis, recursive sequence recombination (e.g., shuffling), oroligonucleotide recombination. In other cases, such polynucleotides canbe made in silico or through oligonucleotide recombination methods asdescribed in the references cited herein.

As described in more detail herein, the polynucleotides of the inventioninclude polynucleotides that encode polypeptides of the invention,polynucleotide sequences complementary to these polynucleotidesequences, and polynucleotides that hybridize under at least stringentconditions to the sequences defined herein. A coding sequence refers toa polynucleotide sequence encoding a particular polypeptide or domain,region, or fragment of said polypeptide. A coding sequence may encode(code for) a polypeptide of the invention exhibiting an interferon alphaactivity as described above. The polynucleotides of the invention may bein the form of RNA or in the form of DNA, and include mRNA, cRNA,synthetic RNA and DNA, and cDNA. The polynucleotides may bedouble-stranded or single-stranded, and if single-stranded, can be thecoding strand or the non-coding (anti-sense, complementary) strand. Thepolynucleotides of the invention include the coding sequence of apolypeptide of the invention (i) in isolation, (ii) in combination withone or more additional coding sequences, so as to encode, e.g., a fusionprotein, a pre-protein, a prepro-protein, or the like, (iii) incombination with non-coding sequences, such as introns, controlelements, such as a promoter (e.g., naturally occurring or recombinantor shuffled promoter), a terminator element, or 5′ and/or 3′untranslated regions effective for expression of the coding sequence ina suitable host, and/or (iv) in a vector, cell, or host environment inwhich the coding sequence is a heterologous gene.

Polynucleotides of the invention can also be found in combination withtypical compositional formulations of nucleic acids, including in thepresence of carriers, buffers, adjuvants, excipients, and the like, asare known to those of ordinary skill in the art. Polynucleotidefragments typically comprise at least about 200 nucleotide bases, suchas at least about 250, 300, 350, 400, 450, 460, 470, or more bases. Thenucleotide fragments of polynucleotides of the invention may hybridizeunder highly stringent conditions to a polynucleotide sequence describedherein and/or encode amino acid sequences having at least one of theproperties of polypeptides of the invention described herein.

Modified Coding Sequences

As will be understood by those of ordinary skill in the art, it can beadvantageous to modify a coding sequence to enhance its expression in aparticular host. The genetic code is redundant with 64 possible codons,but most organisms preferentially use a subset of these codons. Thecodons that are utilized most often in a species are considered optimalcodons, and those not utilized very often are classified as rare orlow-usage codons (see, e.g., Zhang, S. P. et al. (1991) Gene 105:61-72).Codons can be substituted to reflect the preferred codon usage of thehost, a process sometimes termed “codon optimization” or “controllingfor species codon bias.”

Modified coding sequence containing codons preferred by a particularprokaryotic or eukaryotic host (see, e.g., Murray, E. et al. (1989) NucAcids Res 17:477-508) can be prepared, for example, to increase the rateof translation or to produce recombinant RNA transcripts havingdesirable properties, such as a longer half-life, as compared withtranscripts produced from a non-optimized sequence. Translation stopcodons can also be modified to reflect host preference. For example,preferred stop codons for S. cerevisiae and mammals are UAA and UGArespectively. The preferred stop codon for monocotyledonous plants isUGA, whereas insects and E. coli prefer to use UAA as the stop codon(Dalphin, M. E. et al. (1996) Nucl. Acids Res. 24:216-218).

The polynucleotide sequences of the present invention can be engineeredin order to alter a coding sequence of the invention for a variety ofreasons, including but not limited to, alterations which modify thecloning, processing and/or expression of the gene product. For example,alterations may be introduced using techniques which are well known inthe art, e.g., site-directed mutagenesis, to insert new restrictionsites, to alter glycosylation patterns, to introduce or removeattachment groups (e.g., for pegylation or other conjugation), to changecodon preference, to introduce splice sites, etc.

Silent Variations

Because of the degeneracy of the genetic code, a large number offunctionally identical nucleic acids encode any given polypeptide. Forinstance, inspection of the codon table below (Table 5) shows thatcodons AGA, AGG, CGA, CGC, CGG, and CGU all encode the amino acidarginine. Thus, at every position in a nucleic acid sequence where anarginine is specified by a codon, the codon can be altered to any of thecorresponding codons described above without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations”. It isto be understood that U in an RNA sequence corresponds to T in a DNAsequence.

TABLE 5 Codon Table Amino acid Codon(s) Alanine Ala A GCA GCC GCG GCUCysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acid Glu EGAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

It will thus be appreciated by those skilled in the art that due to thedegeneracy of the genetic code, a multitude of nucleic acids sequencesencoding polypeptides of the invention may be produced, some of whichmay bear minimal sequence identity to the nucleic acid sequencesexplicitly disclosed herein. One of ordinary skill in the art willrecognize that each codon in a nucleic acid (except AUG and UGC, whichare ordinarily the only codon for methionine and tryptophan,respectively) can be modified by standard techniques to encode afunctionally identical polypeptide. Accordingly, each silent variationof a nucleic acid which encodes a polypeptide is implicit in anydescribed sequence. The invention also provides each and every possiblevariation of a nucleic acid sequence encoding a polypeptide of theinvention that can be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet (codon) genetic code (e.g., as set forth in Table 5),as applied to the nucleic acid sequence encoding a polypeptide of theinvention. All such variations of every nucleic acid herein arespecifically provided and described by consideration of the sequence incombination with the genetic code. One of skill is fully able togenerate any silent substitution of the sequences listed herein.

Using Polynucleotides

The polynucleotides of the invention have a variety of uses in, forexample, recombinant production (i.e., expression) of the polypeptidesof the invention typically through expression of a plasmid expressionvector comprising a sequence encoding the polypeptide or fragmentthereof; as therapeutics; as prophylactics; as diagnostic tools; asimmunogens; as adjuvants; as diagnostic probes for the presence ofcomplementary or partially complementary nucleic acids (including fordetection of a wild-type interferon-alpha nucleic acid), as substratesfor further reactions, e.g., recursive sequence recombination reactionsor mutation reactions to produce new and/or improved variants, and thelike.

Vectors, Promoters, and Expression Systems

The present invention also includes recombinant constructs comprisingone or more of the nucleic acid sequences as broadly described above.The constructs comprise a vector, such as, a plasmid, a cosmid, a phage,a virus, a bacterial artificial chromosome (BAC), a yeast artificialchromosome (YAC), and the like, into which a nucleic acid sequence ofthe invention has been inserted, in a forward or reverse orientation. Insome instances, the construct further comprises regulatory sequences,including, for example, a promoter, operably linked to the nucleic acidsequence. Large numbers of suitable vectors and promoters are known tothose of skill in the art, and are commercially available.

General texts that describe molecular biological techniques usefulherein, including the use of vectors, promoters and many other relevanttopics, include Berger, supra; Sambrook (1989), supra, and Ausubel,supra. Examples of techniques sufficient to direct persons of skillthrough in vitro amplification methods, including the polymerase chainreaction (PCR) the ligase chain reaction (LCR), Qβ-replicaseamplification and other RNA polymerase mediated techniques (e.g.,NASBA), e.g., for the production of the homologous nucleic acids of theinvention are found in Berger, Sambrook, and Ausubel, all supra, as wellas Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols: A Guideto Methods and Applications (Innis et al., eds.) Academic Press Inc. SanDiego, Calif. (1990) (“Innis”); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3:81-94; (Kwoh et al. (1989)Proc Natl Acad Sci USA 86:1173-1177; Guatelli et al. (1990) Proc NatlAcad Sci USA 87:1874-1878; Lomeli et al. (1989) J Clin Chem35:1826-1831; Landegren et al. (1988) Science 241:1077-1080; Van Brunt(1990) Biotechnology 8:291-294; Wu and Wallace (1989) Gene 4:560-569;Barringer et al. (1990) Gene 89:117-122, and Sooknanan and Malek (1995)Biotechnology 13:563-564. Improved methods of cloning in vitro amplifiednucleic acids are described in Wallace et al., U.S. Pat. No. 5,426,039.Improved methods of amplifying large nucleic acids by PCR are summarizedin Cheng et al. (1994) Nature 369:684-685 and the references therein, inwhich PCR amplicons of up to 40 kilobases (kb) are generated. One ofskill will appreciate that essentially any RNA can be converted into adouble stranded DNA suitable for restriction digestion, PCR expansionand sequencing using reverse transcriptase and a polymerase. SeeAusubel, Sambrook and Berger, all supra.

The present invention also provides host cells that are transduced withvectors of the invention, and the production of polypeptides of theinvention by recombinant techniques. Host cells are geneticallyengineered (e.g., transduced, transformed or transfected) with thevectors of this invention, which may be, for example, a cloning vectoror an expression vector. The vector may be, for example, in the form ofa plasmid, a viral particle, a phage, etc. The engineered host cells canbe cultured in conventional nutrient media modified as appropriate foractivating promoters, selecting transformants, or amplifying genes. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to those skilled in the art and in the references cited herein,including, e.g., Freshney (1994) Culture of Animal Cells, a Manual ofBasic Technique, third edition, Wiley-Liss, New York and the referencescited therein.

The polypeptides of the invention can also be produced in non-animalcells such as plants, yeast, fungi, bacteria and the like. In additionto Sambrook, Berger and Ausubel, details regarding cell culture arefound in, e.g., Payne et al. (1992) Plant Cell and Tissue Culture inLiquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg andPhillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; FundamentalMethods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg N.Y.);Atlas & Parks (eds.) The Handbook of Microbiological Media (1993) CRCPress, Boca Raton, Fla.

The polynucleotides of the present invention and fragments thereof maybe included in any one of a variety of expression vectors for expressinga polypeptide. Such vectors include chromosomal, nonchromosomal andsynthetic DNA sequences, e.g., derivatives of SV40, bacterial plasmids,phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus, pseudorabies, adeno-associated virus,retroviruses and many others. Any vector that transduces geneticmaterial into a cell, and, if replication is desired, which isreplicable and viable in the relevant host can be used.

The nucleic acid sequence in the expression vector is operatively linkedto an appropriate transcription control sequence (promoter) to directmRNA synthesis. Examples of such promoters include: LTR or SV40promoter, E. coli lac or trp promoter, phage lambda P_(L) promoter, CMVpromoter, and other promoters known to control expression of genes inprokaryotic or eukaryotic cells or their viruses. The expression vectoralso contains a ribosome binding site for translation initiation, and atranscription terminator. The vector optionally includes appropriatesequences for amplifying expression, e.g., an enhancer. In addition, theexpression vectors optionally comprise one or more selectable markergenes to provide a phenotypic trait for selection of transformed hostcells, such as dihydrofolate reductase or neomycin resistance foreukaryotic cell culture, or such as tetracycline or ampicillinresistance in E. coli.

The vector containing the appropriate DNA sequence encoding apolypeptide of the invention, as well as an appropriate promoter orcontrol sequence, may be employed to transform an appropriate host topermit the host to express the polypeptide. Examples of appropriateexpression hosts include: bacterial cells, such as E. coli,Streptomyces, and Salmonella typhimurium; fungal cells, such asSaccharomyces cerevisiae, Pichia pastoris, and Neurospora crassa; insectcells such as Drosophila and Spodoptera frugiperda; mammalian cells suchas CHO, COS, BHK, HEK 293 or Bowes melanoma; plant cells, etc. It isunderstood that not all cells or cell lines need to be capable ofproducing fully functional polypeptides of the invention or fragmentsthereof; for example, antigenic fragments of the polypeptide may beproduced in a bacterial or other expression system. The invention is notlimited by the host cells employed.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the polypeptide or fragment thereof.For example, when large quantities of a polypeptide or fragments thereofare needed for the induction of antibodies, vectors which direct highlevel expression of fusion proteins that are readily purified may bedesirable. Such vectors include, but are not limited to, multifunctionalE. coli Cloning and expression vectors such as BLUESCRIPT (Stratagene),in which the nucleotide coding sequence may be ligated into the vectorin-frame with sequences for the amino-terminal Met and the subsequent 7residues of beta-galactosidase so that a hybrid protein is produced; pINvectors (Van Heeke & Schuster (1989) J Biol Chem 264:5503-5509); pETvectors (Novagen, Madison Wis.); and the like.

Similarly, in the yeast Saccharomyces cerevisiae a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase and PGH may be used for production of the polypeptidesof the invention. For reviews, see Ausubel, supra, Berger, supra, andGrant et al. (1987) Methods in Enzymology 153:516-544.

In mammalian host cells, a number of expression systems, such asviral-based systems, may be utilized. In cases where an adenovirus isused as an expression vector, a coding sequence is optionally ligatedinto an adenovirus transcription/translation complex consisting of thelate promoter and tripartite leader sequence. Insertion in anonessential E1 or E3 region of the viral genome results in a viablevirus capable of expressing a polypeptide of the invention in infectedhost cells (Logan and Shenk (1984) Proc Natl Acad Sci USA 81:3655-3659).In addition, transcription enhancers, such as the rous sarcoma virus(RSV) enhancer, are used to increase expression in mammalian host cells.Host cells, media, expression systems, and methods of production includethose known for cloning and expression of various mammalianinterferon-alphas (e.g., human interferon-alphas).

Additional Expression Elements

Specific initiation signals can aid in efficient translation of apolynucleotide coding sequence of the invention and/or fragmentsthereof. These signals can include, e.g., the ATG initiation codon andadjacent sequences. In cases where an coding sequence, its initiationcodon and upstream sequences are inserted into the appropriateexpression vector, no additional translational control signals may beneeded. However, in cases where only coding sequence (e.g., a matureprotein coding sequence), or a portion thereof, is inserted, exogenousnucleic acid transcriptional control signals including the ATGinitiation codon must be provided. Furthermore, the initiation codonmust be in the correct reading frame to ensure transcription of theentire insert. Exogenous transcriptional elements and initiation codonscan be of various origins, both natural and synthetic. The efficiency ofexpression can enhanced by the inclusion of enhancers appropriate to thecell system in use (see, e.g., Scharf D. et al. (1994) Results ProblCell Differ 20:125-62; and Bittner et al. (1987) Methods in Enzymol153:516-544).

Secretion/Localization Sequences

Polynucleotides encoding polypeptides of the invention can also befused, for example, in-frame to nucleic acid encoding asecretion/localization sequence, to target polypeptide expression to adesired cellular compartment, membrane, or organelle, or to directpolypeptide secretion to the periplasmic space or into the cell culturemedia. Such sequences are known to those of skill, and include secretionleader or signal peptides, organelle targeting sequences (e.g., nuclearlocalization sequences, ER retention signals, mitochondrial transitsequences, chloroplast transit sequences), membrane localization/anchorsequences (e.g., stop transfer sequences, GPI anchor sequences), and thelike.

Expression Hosts

In a further aspect, the present invention relates to host cellscontaining any of the above-described nucleic acids, vectors, or otherconstructs of the invention. The host cell can be a eukaryotic cell,such as a mammalian cell, a yeast cell, or a plant cell, or the hostcell can be a prokaryotic cell, such as a bacterial cell. Introductionof the construct into the host cell can be effected by calcium phosphatetransfection, DEAE-Dextran mediated transfection, electroporation, geneor vaccine gun, injection, or other common techniques (see, e.g., Davis,L., Dibner, M., and Battey, I. (1986) Basic Methods in MolecularBiology) for in vivo, ex vivo or in vitro methods.

A host cell strain is optionally chosen for its ability to modulate theexpression of the inserted sequences or to process the expressed proteinin the desired fashion. Such modifications of the protein include, butare not limited to, acetylation, carboxylation, glycosylation,phosphorylation, lipidation and acylation. Post-translational processingwhich cleaves a “pre” or a “prepro” form of the protein may also beimportant for correct insertion, folding and/or function. Different hostcells such as E. coli, Bacillus sp., yeast or mammalian cells such asCHO, HeLa, BHK, MDCK, HEK 293, WI38, etc. have specific cellularmachinery and characteristic mechanisms for such post-translationalactivities and may be chosen to ensure the correct modification andprocessing of the introduced foreign protein.

Stable expression can be used for long-term, high-yield production ofrecombinant proteins. For example, cell lines which stably express apolypeptide of the invention are transduced using expression vectorswhich contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells may be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth and recovery of cells which successfully express theintroduced sequences. For example, resistant clumps of stablytransformed cells can be proliferated using tissue culture techniquesappropriate to the cell type.

Host cells transformed with a nucleotide sequence encoding a polypeptideof the invention are optionally cultured under conditions suitable forthe expression and recovery of the encoded protein from cell culture.The polypeptide produced by a recombinant cell may be secreted,membrane-bound, or contained intracellularly, depending on the sequenceand/or the vector used. As will be understood by those of skill in theart, expression vectors containing polynucleotides encoding polypeptidesof the invention can be designed with signal sequences which directsecretion of the mature polypeptides through a prokaryotic or eukaryoticcell membrane.

Additional Sequences

The polynucleotides of the present invention optionally comprise acoding sequence fused in-frame to a marker sequence which, e.g.,facilitates purification and/or detection of the encoded polypeptide.Such purification subsequences include, but are not limited to, metalchelating peptides such as histidine-tryptophan modules that allowpurification on immobilized metals, a sequence which binds glutathione(e.g., GST), a hemagglutinin (HA) tag (corresponding to an epitopederived from the influenza hemagglutinin protein; Wilson, I. et al.(1984) Cell 37:767), maltose binding protein sequences, the FLAG epitopeutilized in the FLAGS extension/affinity purification system, and thelike. The inclusion of a protease-cleavable polypeptide linker sequencebetween the purification domain and the polypeptide sequence is usefulto facilitate purification.

For example, one expression vector possible to use in the compositionsand methods described herein provides for expression of a fusion proteincomprising a polypeptide of the invention fused to a polyhistidineregion separated by an enterokinase cleavage site. The histidineresidues facilitate purification on IMIAC (immobilized metal ionaffinity chromatography, as described in Porath et al. (1992) ProteinExpression and Purification 3:263-281) while the enterokinase cleavagesite provides a method for separating the desired polypeptide from thepolyhistidine region. pGEX vectors (Promega; Madison, Wis.) areoptionally used to express foreign polypeptides as fusion proteins withglutathione S-transferase (GST). In general, such fusion proteins aresoluble and can easily be purified from lysed cells by adsorption toligand-agarose beads (e.g., glutathione-agarose in the case ofGST-fusions) followed by elution in the presence of free ligand.

An additional construction in the compositions and methods describedherein provides for proteins, and their encoding nucleic acids,comprising polypeptides of the invention (or one or more fragmentsthereof), e.g., as described herein, fused to an Ig molecule, e.g.,human IgG Fc (“fragment crystallizable,” or fragment complement binding)hinge, CH2 domain and CH3 domain (and nucleotide sequences encodingthem). Fc is the portion of the antibody responsible for binding toantibody receptors on cells and the C1q component of complement. Thesefusion proteins or fragments thereof and their encoding nucleic acidsare optionally useful as prophylactic and/or therapeutic drugs or asdiagnostic tools (see also, e.g., Challita-Eid, P. et al. (1998) JImmunol 160:3419-3426; Sturmhoefel, K. et al. (1999) Cancer Res59:4964-4972).

Polypeptide Production and Recovery

Following transduction of a suitable host strain and growth of the hoststrain to an appropriate cell density, the selected promoter is inducedby appropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. Cells are typicallyharvested by centrifugation, disrupted by physical or chemical means,and the resulting crude extract retained for further purification.Eukaryotic or microbial cells employed in expression of the proteins canbe disrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents, orother methods, which are well know to those skilled in the art.

As noted, many references are available for the culture and productionof many cells, including cells of bacterial, plant, animal (especiallymammalian) and archebacterial origin. See, e.g., Sambrook, Ausubel, andBerger (all supra), as well as Freshney (1994) Culture of Animal Cells,a Manual of Basic Technique, third edition, Wiley-Liss, New York and thereferences cited therein; Doyle and Griffiths (1997) Mammalian CellCulture: Essential Techniques John Wiley and Sons, NY; Humason (1979)Animal Tissue Techniques, fourth edition W.H. Freeman and Company; andRicciardelli et al. (1989) In vitro Cell Dev Biol 25:1016-1024. Forplant cell culture and regeneration see, e.g., Payne et al. (1992) PlantCell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. NewYork, N.Y.; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue andOrgan Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin Heidelberg New York) and Plant Molecular Biology (1993) R. R. D.Croy (ed.) Bios Scientific Publishers, Oxford, U.K. ISBN 0 12 198370 6.Cell culture media in general are set forth in Atlas and Parks (eds.)The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.Additional information for cell culture is found in available commercialliterature such as the Life Science Research Cell Culture Catalogue fromSigma-Aldrich, Inc (St Louis, Mo.) (“Sigma-LSRCCC”) and, e.g., the PlantCulture Catalogue and supplement also from Sigma-Aldrich, Inc (St Louis,Mo.) (“Sigma-PCCS”).

Polypeptides of the invention can be recovered and purified fromrecombinant cell cultures by any of a number of methods well known inthe art, including ammonium sulfate or ethanol precipitation, acidextraction, anion or cation exchange chromatography, phosphocellulosechromatography, hydrophobic interaction chromatography, affinitychromatography (e.g., using any of the tagging systems noted herein),hydroxylapatite chromatography, and lectin chromatography. Proteinrefolding steps can be used, as desired, in completing configuration ofthe mature protein or fragments thereof. Finally, high performanceliquid chromatography (HPLC) can be employed in the final purificationsteps. In addition to the references noted, supra, a variety ofpurification methods are well known in the art, including, e.g., thoseset forth in Sandana (1997) Bioseparation of Proteins, Academic Press,Inc.; Bollag et al. (1996) Protein Methods, 2^(nd) Edition Wiley-Liss,NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ;Harris and Angal (1990) Protein Purification Applications: A PracticalApproach IRL Press at Oxford, Oxford, England; Harris and Angal ProteinPurification Methods: A Practical Approach IRL Press at Oxford, Oxford,England; Scopes (1993) Protein Purification: Principles and Practice3^(rd) Edition Springer Verlag, NY; Janson and Ryden (1998) ProteinPurification: Principles, High Resolution Methods and Applications,Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols onCD-ROM Humana Press, NJ.

In Vitro Expression Systems

Cell-free transcription/translation systems can also be employed toproduce polypeptides of the invention using polynucleotides of thepresent invention. Several such systems are commercially available. Ageneral guide to in vitro transcription and translation protocols isfound in Tymms (1995) In vitro Transcription and Translation Protocols:Methods in Molecular Biology Volume 37, Garland Publishing, NY.

In Vivo Uses and Applications

Polynucleotides that encode a polypeptide of the invention, orcomplements of the polynucleotides (including e.g., antisense orribozyme molecules), are optionally administered to a cell to accomplisha therapeutically useful process or to express a therapeutically usefulproduct. These in vivo applications, including gene therapy, include amultitude of techniques by which gene expression may be altered incells. Such methods include, for instance, the introduction of genes forexpression of, e.g., therapeutically and/or prophylactically usefulpolypeptides, such as the polypeptides of the present invention.

In Vivo Polypeptide Expression

Polynucleotides encoding polypeptides of the invention are particularlyuseful for in vivo therapeutic applications, using techniques well knownto those skilled in the art. For example, cultured cells are engineeredex vivo with at least one polynucleotide (DNA or RNA) of the inventionand/or other polynucleotide sequences encoding, e.g., at least one of anantigen, cytokine, other co-stimulatory molecule, adjuvant, etc., andthe like, with the engineered cells then being returned to the patient.Cells may also be engineered in vivo for expression of one or morepolypeptides in vivo. including polypeptides and/or antigenic peptidesof the invention.

A number of viral vectors suitable for organismal in vivo transductionand expression are known. Such vectors include retroviral vectors (see,e.g., Miller, Curr Top Microbiol Immunol (1992) 158:1-24; Salmons andGunzburg (1993) Human Gene Therapy 4:129-141; Miller et al. (1994)Methods in Enzymology 217:581-599) and adeno-associated vectors(reviewed in Carter (1992) Curr Opinion Biotech 3:533-539; Muzcyzka(1992) Curr Top Microbiol Immunol. 158:97-129). Other viral vectors thatare used include adenoviral vectors, herpes viral vectors and Sindbisviral vectors, as generally described in, e.g., Jolly (1994) Cancer GeneTherapy 1:51-64; Latchman (1994) Molec Biotechnol 2:179-195; andJohanning et al. (1995) Nucl Acids Res 23:1495-1501.

In one aspect, a pox virus vector can be used. The pox viral vector istransfected with a polynucleotide sequence encoding a polypeptide of theinvention, and is useful in prophylactic, therapeutic and diagnosticapplications where enhancement of an immune response, such as e.g.,increased or improved T cell proliferation is desired. See viral vectorsdiscussed in, e.g., Berencsi et al., J Infect Dis (2001)183(8):1171-9;Rosenwirth et al., Vaccine 2001 Feb. 8; 19(13-14):1661-70; Kittlesen etal., J Immunol (2000) 164(8):4204-11; Brown et al. Gene Ther 20007(19):1680-9; Kanesa-thasan et al., Vaccine (2000) 19(4-5):483-91; Sten(2000) Drug 60(2):249-71. Compositions comprising such vectors and anacceptable excipient are also a feature of the invention.

Gene therapy and genetic vaccines provide methods for combating chronicinfectious diseases (e.g., HIV infection, viral hepatitis), as well asnon-infectious diseases including cancer and some forms of congenitaldefects such as enzyme deficiencies, and such methods can be employedwith polynucleotides of the invention, including, e.g., vectors andcells comprising such polynucleotides. Several approaches forintroducing nucleic acids and vectors into cells in vivo, ex vivo and invitro have been used and can be employed with polynucleotides of theinvention, and vectors comprising such polynucleotides. These approachesinclude liposome based gene delivery (Debs and Zhu (1993) WO 93/24640and U.S. Pat. No. 5,641,662; Mannino and Gould-Fogerite (1988)BioTechniques 6(7):682-691; Rose, U.S. Pat. No. 5,279,833; Brigham(1991) WO 91/06309; and Felgner et al. (1987) Proc Natl Acad Sci USA84:7413-7414; Brigham et al. (1989) Am J Med Sci 298:278-281; Nabel etal. (1990) Science 249:1285-1288; Hazinski et al. (1991) Am J Resp CellMolec Biol 4:206-209; and Wang and Huang (1987) Proc Natl Acad Sci USA84:7851-7855); adenoviral vector mediated gene delivery, e.g., to treatcancer (see, e.g., Chen et al. (1994) Proc Natl Acad Sci USA91:3054-3057; Tong et al. (1996) Gynecol Oncol 61:175-179; Clayman etal. (1995) Cancer Res. 5:1-6; O'Malley et al. (1995) Cancer Res55:1080-1085; Hwang et al. (1995) Am J Respir Cell Mol Biol 13:7-16;Haddada et al. (1995) Curr Top Microbiol Immunol. 1995 (Pt. 3):297-306;Addison et al. (1995) Proc Natl Acad Sci USA 92:8522-8526; Colak et al.(1995) Brain Res 691:76-82; Crystal (1995) Science 270:404-410; Elshamiet al. (1996) Human Gene Ther 7:141-148; Vincent et al. (1996) JNeurosurg 85:648-654), and many others. Replication-defective retroviralvectors harboring therapeutic polynucleotide sequence as part of theretroviral genome have also been used, particularly with regard tosimple MuLV vectors. See, e.g., Miller et al. (1990) Mol Cell Biol10:4239 (1990); Kolberg (1992) J NIH Res 4:43, and Cornetta et al.(1991) Hum Gene Ther 2:215). Nucleic acid transport coupled toligand-specific, cation-based transport systems (Wu and Wu (1988) J BiolChem, 263:14621-14624) has also been used. Naked DNA expression vectorshave also been described (Nabel et al. (1990), supra); Wolff et al.(1990) Science, 247:1465-1468). In general, these approaches can beadapted to the invention by incorporating nucleic acids encoding thepolypeptides of the invention into the appropriate vectors.

General texts which describe gene therapy protocols, which can beadapted to the present invention by introducing the nucleic acids of theinvention into patients, include, e.g., Robbins (1996) Gene TherapyProtocols, Humana Press, NJ, and Joyner (1993) Gene Targeting: APractical Approach, IRL Press, Oxford, England.

Antisense Technology

In addition to expression of the nucleic acids of the invention as genereplacement nucleic acids, the nucleic acids are also useful for senseand anti-sense suppression of expression, e.g., to down-regulateexpression of a nucleic acid of the invention, once, or when, expressionof the nucleic acid is no-longer desired in the cell. Similarly, thenucleic acids of the invention, or subsequences or anti-sense sequencesthereof, can also be used to block expression of naturally occurringhomologous nucleic acids. A variety of sense and anti-sense technologiesare known in the art, e.g., as set forth in Lichtenstein and Nellen(1997) Antisense Technology: A Practical Approach IRL Press at OxfordUniversity, Oxford, England, and in Agrawal (1996) AntisenseTherapeutics Humana Press, NJ, and the references cited therein.

Use as Probes

Also contemplated are uses of polynucleotides, also referred to hereinas oligonucleotides, typically having at least 12 bases, preferably atleast 15, more preferably at least 20, at least 30, or at least 50 ormore bases, which hybridize under highly stringent conditions to apolynucleotide of the invention, or fragments thereof. Thepolynucleotides may be used as probes, primers, sense and antisenseagents, and the like, according to methods as noted supra.

Nucleic Acid Hybridization

Nucleic acids “hybridize” when they associate, typically in solution.Nucleic acids hybridize due to a variety of well characterizedphysico-chemical forces, such as hydrogen bonding, solvent exclusion,base stacking and the like. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, part I, chapter 2, “Overview of principles of hybridization andthe strategy of nucleic acid probe assays,” (Elsevier, New York)(hereinafter “Tjissen”), as well as in Ausubel, supra, Hames and Higgins(1995) Gene Probes 1, IRL Press at Oxford University Press, Oxford,England (Hames and Higgins 1) and Hames and Higgins (1995) Gene Probes2, IRL Press at Oxford University Press, Oxford, England (Hames andHiggins 2) provide details on the synthesis, labeling, detection andquantification of DNA and RNA, including oligonucleotides.

An indication that two nucleic acid sequences are substantiallyidentical is that the two molecules hybridize to each other under atleast stringent conditions. The phrase “hybridizing specifically to,”refers to the binding, duplexing, or hybridizing of a molecule only to aparticular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetpolynucleotide sequence.

“Stringent hybridization wash conditions” and “stringent hybridizationconditions” in the context of nucleic acid hybridization experiments,such as Southern and northern hybridizations, are sequence dependent,and are different under different environmental parameters. An extensiveguide to hybridization of nucleic acids is found in Tijssen (1993),supra, and in Hames and Higgins 1 and Hames and Higgins 2, supra.

For purposes of the present invention, generally, “highly stringent”hybridization and wash conditions are selected to be about 5° C. or lesslower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength and pH (as noted below, highly stringentconditions can also be referred to in comparative terms). The T_(m) isthe temperature (under defined ionic strength and pH) at which 50% ofthe test sequence hybridizes to a perfectly matched probe. In otherwords, the T_(m) indicates the temperature at which the nucleic acidduplex is 50% denatured under the given conditions and its represents adirect measure of the stability of the nucleic acid hybrid. Thus, theT_(m) corresponds to the temperature corresponding to the midpoint intransition from helix to random coil; it depends on length, nucleotidecomposition, and ionic strength for long stretches of nucleotides.Typically, under “stringent conditions,” a probe will hybridize to itstarget subsequence, but to no other sequences. “Very stringentconditions” are selected to be equal to the T_(m) for a particularprobe.

After hybridization, unhybridized nucleic acid material can be removedby a series of washes, the stringency of which can be adjusted dependingupon the desired results. Low stringency washing conditions (e.g., usinghigher salt and lower temperature) increase sensitivity, but can productnonspecific hybridization signals and high background signals. Higherstringency conditions (e.g., using lower salt and higher temperaturethat is closer to the hybridization temperature) lowers the backgroundsignal, typically with only the specific signal remaining. See, Rapley,R. and Walker, J. M. eds., Molecular Biomethods Handbook (Humana Press,Inc. 1998) (hereinafter “Rapley and Walker”), which is incorporatedherein by reference in its entirety for all purposes.

The T_(m) of a DNA-DNA duplex can be estimated using equation (1):T _(m)(° C.)=81.5° C.+16.6(log₁₀ M)+0.41(%G+C)−0.72 (%f)−500/n,where M is the molarity of the monovalent cations (usually Na+), (% G+C)is the percentage of guanosine (G) and cystosine (C) nucleotides, (% f)is the percentage of formalize and n is the number of nucleotide bases(i.e., length) of the hybrid. See, Rapley and Walker, supra.

The T_(m) of an RNA-DNA duplex can be estimated using equation (2):T _(m)(° C.)=79.8° C.+18.5(log₁₀M)+0.58(%G+C)−11.8(%G+C)−0.56(%f)−820/n,where M is the molarity of the monovalent cations (usually Na+), (% G+C)is the percentage of guanosine (G) and cystosine (C) nucleotides, (% f)is the percentage of formamide and n is the number of nucleotide bases(i.e., length) of the hybrid. Id. Equations 1 and 2 above are typicallyaccurate only for hybrid duplexes longer than about 100-200 nucleotides.Id.

The Tm of nucleic acid sequences shorter than 50 nucleotides can becalculated as follows:

T_(m) (° C.)=4(G+C)+2(A+T), where A (adenine), C, T (thymine), and G arethe numbers of the corresponding nucleotides.

An example of stringent hybridization conditions for hybridization ofcomplementary nucleic acids which have more than 100 Complementaryresidues on a filter in a Southern or northern blot is 50% formalin (orformamide) with 1 mg of heparin at 42° C., with the hybridization beingcarried out overnight. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see Sambrook, supra, for adescription of SSC buffer). Often, the high stringency wash is precededby a low stringency wash to remove background probe signal. An examplelow stringency wash is 2×SSC at 40° C. for 15 minutes. An example ofhighly stringent wash conditions is 0.15M NaCl at 72° C. for about 15minutes. An example medium stringency wash for a duplex of, e.g., morethan 100 nucleotides, is 1×SSC at 45° C. for 15 minutes. An example lowstringency wash for a duplex of, e.g., more than 100 nucleotides, is4-6×SSC at 40° C. for 15 minutes. For short probes (e.g., about 10 to 50nucleotides), stringent conditions typically involve salt concentrationsof less than about 1.0 M Na⁺ ion, typically about 0.01 to 1.0 M Na⁺ ionconcentration (or other salts) at pH 7.0 to 8.3, and the temperature istypically at least about 30° C. Stringent conditions can also beachieved with the addition of destabilizing agents such as formamide.

In general, a signal to noise ratio of 2× or 2.5×-5× (or higher) thanthat observed for an unrelated probe in the particular hybridizationassay indicates detection of a specific hybridization. Detection of atleast stringent hybridization between two sequences in the context ofthe present invention indicates relatively strong structural similarityor homology to, e.g., the nucleic acids of the present inventionprovided in the sequence listings herein.

As noted, “highly stringent” conditions are selected to be about 5° C.or less lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. Target sequences that areclosely related or identical to the nucleotide sequence of interest(e.g., “probe”) can be identified under highly stringency conditions.Lower stringency conditions are appropriate for sequences that are lesscomplementary. See, e.g., Rapley and Walker; Sambrook, all supra.

Comparative hybridization can be used to identify nucleic acids of theinvention, and this comparative hybridization method is a preferredmethod of distinguishing nucleic acids of the invention. Detection ofhighly stringent hybridization between two nucleotide sequences in thecontext of the present invention indicates relatively strong structuralsimilarity/homology to, e.g., the nucleic acids provided in the sequencelisting herein. Highly stringent hybridization between two nucleotidesequences demonstrates a degree of similarity or homology of structure,nucleotide base composition, arrangement or order that is greater thanthat detected by stringent hybridization conditions. In particular,detection of highly stringent hybridization in the context of thepresent invention indicates strong structural similarity or structuralhomology (e.g., nucleotide structure, base composition, arrangement ororder) to, e.g., the nucleic acids provided in the sequence listingsherein. For example, it is desirable to identify test nucleic acidswhich hybridize to the exemplar nucleic acids herein under stringentconditions.

Thus, one measure of stringent hybridization is the ability to hybridizeto one of the listed nucleic acids of the invention (e.g., nucleic acidsequences SEQ ID NOS:16-30, and complementary polynucleotide sequencesthereof) under highly stringent conditions (or very stringentconditions, or ultra-high stringency hybridization conditions, orultra-ultra high stringency hybridization conditions). Stringenthybridization (including, e.g., highly stringent, ultra-high stringency,or ultra-ultra high stringency hybridization conditions) and washconditions can easily be determined empirically for any test nucleicacid.

For example, in determining highly stringent hybridization and washconditions, the hybridization and wash conditions are graduallyincreased (e.g., by increasing temperature, decreasing saltconcentration, increasing detergent concentration and/or increasing theconcentration of organic solvents, such as formalin, in thehybridization or wash), until a selected set of criteria are met. Forexample, the hybridization and wash conditions are gradually increaseduntil a probe comprising one or more nucleic acid sequences selectedfrom SEQ ID NOS:16-30, and complementary polynucleotide sequencesthereof, binds to a perfectly matched complementary target (again, anucleic acid comprising one or more nucleic acid sequences selected fromSEQ ID NOS: 16-30, and complementary polynucleotide sequences thereof),with a signal to noise ratio that is at least 2.5×, and optionally 5× ormore as high as that observed for hybridization of the probe to anunmatched target. In this case, the unmatched target is a nucleic acidcorresponding to, e.g., a known interferon-alpha nucleic acid sequence(e.g., an interferon-alpha nucleic acid sequence present in a publicdatabase such as GenBank or GENESEQ at the time of filing of the subjectapplication).

A test nucleic acid is said to specifically hybridize to a probe nucleicacid when it hybridizes at least ½ as well to the probe as to theperfectly matched complementary target, i.e., with a signal to noiseratio at least ½ as high as hybridization of the probe to the targetunder conditions in which the perfectly matched probe binds to theperfectly matched complementary target with a signal to noise ratio thatis at least about 2.5×-10×, typically 5×-10× as high as that observedfor hybridization to any of the unmatched target nucleic acids such as,e.g., a known interferon-alpha nucleic acid sequence as set forth above.For some such nucleic acids, the stringent conditions are selected suchthat a perfectly complementary oligonucleotide to the codingoligonucleotide hybridizes to the coding oligonucleotide with at leastabout a 5× higher signal to noise ratio than for hybridization of theperfectly complementary oligonucleotide to a control nucleic acidcorresponding to a known interferon-alpha sequence as set forth above.

Ultra high-stringency hybridization and wash conditions are those inwhich the stringency of hybridization and wash conditions are increaseduntil the signal to noise ratio for binding of the probe to theperfectly matched complementary target nucleic acid is at least 10× ashigh as that observed for hybridization to any of the unmatched targetnucleic acids, such as, e.g., a known interferon-alpha nucleic acidsequence as set forth above. A target nucleic acid which hybridizes to aprobe under such conditions, with a signal to noise ratio of at least ½that of the perfectly matched complementary target nucleic acid is saidto bind to the probe under ultra-high stringency conditions.

Similarly, even higher levels of stringency can be determined bygradually increasing the hybridization and/or wash conditions of therelevant hybridization assay. For example, those in which the stringencyof hybridization and wash conditions are increased until the signal tonoise ratio for binding of the probe to the perfectly matchedcomplementary target nucleic acid is at least 10×, 20×, 50×, 100×, or500× or more as high as that observed for hybridization to any of theunmatched target nucleic acids, such as, e.g., a known interferon-alphanucleic acid sequence as set forth above. A target nucleic acid whichhybridizes to a probe under such conditions, with a signal to noiseratio of at least ½ that of the perfectly matched complementary targetnucleic acid is said to bind to the probe under ultra-ultra-highstringency conditions.

Target nucleic acids which hybridize to the nucleic acids represented bySEQ ID NOS: 16-30 under high, ultra-high and ultra-ultra high stringencyconditions are a feature of the invention. Examples of such nucleicacids include those with one or a few silent or conservative nucleicacid substitutions as compared to a given nucleic acid sequence.

Substrates and Formats for Sequence Recombination

The polynucleotides of the invention and fragments thereof areoptionally used as substrates for any of a variety of recombination andrecursive sequence recombination reactions, in addition to their use instandard cloning methods as set forth in, e.g., Ausubel, Berger andSambrook, e.g., to produce additional polynucleotides that encodepolypeptides having desired properties. A variety of such reactions areknown, including those developed by the inventors and their co-workers.

A variety of diversity generating protocols for generating andidentifying molecules having one of more of the properties describedherein are available and described in the art. The procedures can beused separately, and/or in combination to produce one or more variantsof a nucleic acid or set of nucleic acids, as well variants of encodedproteins. Individually and collectively, these procedures providerobust, widely applicable ways of generating diversified nucleic acidsand sets of nucleic acids (including, e.g., nucleic acid libraries)useful, e.g., for the engineering or rapid evolution of nucleic acids,proteins, pathways, cells and/or organisms with new and/or improvedcharacteristics. While distinctions and classifications are made in thecourse of the ensuing discussion for clarity, it will be appreciatedthat the techniques are often not mutually exclusive. Indeed, thevarious methods can be used singly or in combination, in parallel or inseries, to access diverse sequence variants.

The result of any of the diversity generating procedures describedherein can be the generation of one or more nucleic acids, which can beselected or screened for nucleic acids with or which confer desirableproperties, or that encode proteins with or which confer desirableproperties. Following diversification by one or more of the methodsherein, or otherwise available to one of skill, any nucleic acids thatare produced can be selected for a desired activity or property, e.g.,altered binding affinity for an interferon-alpha receptor, alteredantiviral or antiproliferative activity, altered capacities to induceT_(H)1 differentiation, altered abilities to induce or inhibit cytokineproduction. This can include identifying any activity that can bedetected, for example, in an automated or automatable format, by any ofthe assays in the art and the assays of the invention discussed here andin the Example section below. A variety of related (or even unrelated)properties can be evaluated, in serial or in parallel, at the discretionof the practitioner.

Descriptions of a variety of diversity generating procedures forgenerating modified nucleic acid sequences that encode polypeptides asdescribed herein are found in the following publications and thereferences cited therein: Soong, N. et al. (2000) “Molecular breeding ofviruses” Nat Genet. 25(4):436-439; Stemmer, et al. (1999) “Molecularbreeding of viruses for targeting and other clinical properties” TumorTargeting 4:1-4; Ness et al. (1999) “DNA Shuffling of subgenomicsequences of subtilisin” Nature Biotechnology 17:893-896; Chang et al.(1999) “Evolution of a cytokine using DNA family shuffling” NatureBiotechnology 17:793-797; Minshull and Stemmer (1999) “Protein evolutionby molecular breeding” Current Opinion in Chemical Biology 3:284-290;Christians et al. (1999) “Directed evolution of thymidine kinase for AZTphosphorylation using DNA family shuffling” Nature Biotechnology17:259-264; Crameri et al. (1998) “DNA shuffling of a family of genesfrom diverse species accelerates directed evolution” Nature 391:288-291;Crameri et al. (1997) “Molecular evolution of an arsenate detoxificationpathway by DNA shuffling,” Nature Biotechnology 15:436-438; Zhang et al.(1997) “Directed evolution of an effective fucosidase from agalactosidase by DNA shuffling and screening” Proc. Natl. Acad. Sci. USA94:4504-4509; Patten et al. (1997) “Applications of DNA Shuffling toPharmaceuticals and Vaccines” Current Opinion in Biotechnology8:724-733; Crameri et al. (1996) “Construction and evolution ofantibody-phage libraries by DNA shuffling” Nature Medicine 2:100-103;Crameri et al. (1996) “Improved green fluorescent protein by molecularevolution using DNA shuffling” Nature Biotechnology 14:315-319; Gates etal. (1996) “Affinity selective isolation of ligands from peptidelibraries through display on a lac repressor ‘headpiece dimer’” Journalof Molecular Biology 255:373-386; Stemmer (1996) “Sexual PCR andAssembly PCR” In: The Encyclopedia of Molecular Biology. VCH Publishers,New York. pp. 447-457; Crameri and Stemmer (1995) “Combinatorialmultiple cassette mutagenesis creates all the permutations of mutant andwildtype cassettes” BioTechniques 18:194-195; Stemmer et al., (1995)“Single-step assembly of a gene and entire plasmid form large numbers ofoligodeoxy-ribonucleotides” Gene, 164:49-53; Stemmer (1995) “TheEvolution of Molecular Computation” Science 270: 1510; Stemmer (1995)“Searching Sequence Space” Bio/Technology 13:549-553; Stemmer (1994)“Rapid evolution of a protein in vitro by DNA shuffling” Nature370:389-391; and Stemmer (1994) “DNA shuffling by random fragmentationand reassembly: In vitro recombination for molecular evolution.” Proc.Natl. Acad. Sci. USA 91:10747-10751.

The term “shuffling” is used herein to indicate recombination betweennon-identical sequences, in some instances shuffling may includecrossover via homologous recombination or via non-homologousrecombination, such as via cre/lox and/or flp/frt systems. Shuffling canbe carried out by employing a variety of different formats, includingfor example, in vitro and in vivo shuffling formats, in silico shufflingformats, shuffling formats that utilize either double-stranded orsingle-stranded templates, primer based shuffling formats, nucleic acidfragmentation-based shuffling formats, and oligonucleotide-mediatedshuffling formats, all of which are based on recombination eventsbetween non-identical sequences and are described in more detail orreferenced herein below, as well as other similar recombination-basedformats.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller & Smith (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller & Smith (1987)“Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Tayloret al. (1985) “The use of phosphorothioate-modified DNA in restrictionenzyme reactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764;Taylor et al. (1985) “The rapid generation of oligonucleotide-directedmutations at high frequency using phosphorothioate-modified DNA” Nucl.Acids Res. 13: 8765-8787 (1985); Nakamaye & Eckstein (1986) “Inhibitionof restriction endonuclease Nci I cleavage by phosphorothioate groupsand its application to oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 14: 9679-9698; Sayers et al. (1988) “Y-T Exonucleases inphosphorothioate-based oligonucleotide-directed mutagenesis” Nucl. AcidsRes. 16:791-802; and Sayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer et al. (1988) “Improvedenzymatic in vitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz et al. (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional suitable methods include point mismatch repair (Kramer et al.(1984) “Point Mismatch Repair” Cell 38:879-887), mutagenesis usingrepair-deficient host strains (Carter et al. (1985) “Improvedoligonucleotide site-directed mutagenesis using M13 vectors” Nucl. AcidsRes. 13: 4431-4443; and Carter (1987) “Improved oligonucleotide-directedmutagenesis using M13 vectors” Methods in Enzymol. 154: 382-403),deletion mutagenesis (Eghtedarzadeh & Henikoff (1986) “Use ofoligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-purification (Wells et al.(1986) “Importance of hydrogen-bond formation in stabilizing thetransition state of subtilisin” Phil. Trans. R. Soc. Lond. A 317:415-423), mutagenesis by total gene synthesis (Nambiar et al. (1984)“Total synthesis and cloning of a gene coding for the ribonuclease Sprotein” Science 223: 1299-1301; Sakamar and Khorana (1988) “Totalsynthesis and expression of a gene for the a-subunit of bovine rod outersegment guanine nucleotide-binding protein (transducin)” Nucl. AcidsRes. 14: 6361-6372; Wells et al. (1985) “Cassette mutagenesis: anefficient method for generation of multiple mutations at defined sites”Gene 34:315-323; and Grundstrom et al. (1985) “Oligonucleotide-directedmutagenesis by microscale ‘shot-gun’ gene synthesis” Nucl. Acids Res.13: 3305-3316), double-strand break repair (Mandecki (1986)“Oligonucleotide-directed double-strand break repair in plasmids ofEscherichia coli: a method for site-specific mutagenesis” Proc. Natl.Acad. Sci. USA, 83:7177-7181; and Arnold (1993) “Protein engineering forunusual environments” Current Opinion in Biotechnology 4:450-455).Additional details on many of the above methods can be found in Methodsin Enzymology Volume 154, which also describes useful controls fortrouble-shooting problems with various mutagenesis methods.

Additional details regarding various diversity generating methods can befound in the following U.S. patents, PCT publications and applications,and EPO publications: U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25,1997), “Methods for In vitro Recombination;” U.S. Pat. No. 5,811,238 toStemmer et al. (Sep. 22, 1998) “Methods for Generating Polynucleotideshaving Desired Characteristics by Iterative Selection andRecombination;” U.S. Pat. No. 5,830,721 to Stemmer et al. (Nov. 3,1998), “DNA Mutagenesis by Random Fragmentation and Reassembly;” U.S.Pat. No. 5,834,252 to Stemmer, et al. (Nov. 10, 1998) “End-ComplementaryPolymerase Reaction;” U.S. Pat. No. 5,837,458 to Minshull, et al. (Nov.17, 1998), “Methods and Compositions for Cellular and MetabolicEngineering;” WO 95/22625, Stemmer and Crameri, “Mutagenesis by RandomFragmentation and Reassembly;” WO 96/33207 by Stemmer and Lipschutz “EndComplementary Polymerase Chain Reaction;” WO 97/20078 by Stemmer andCrameri “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” WO 97/35966by Minshull and Stemmer, “Methods and Compositions for Cellular andMetabolic Engineering;” WO 99/41402 by Punnonen et al. “Targeting ofGenetic Vaccine Vectors;” WO 99/41383 by Punnonen et al. “AntigenLibrary Immunization;” WO 99/41369 by Punnonen et al. “Genetic VaccineVector Engineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in vitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination;” WO 00/18906 by Patten etal., “Shuffling of Codon-Altered Genes;” WO 00/04190 by del Cardayre etal. “Evolution of Whole Cells and Organisms by Recursive Recombination;”WO 00/42561 by Crameri et al., “Oligonucleotide Mediated Nucleic AcidRecombination;” WO 00/42559 by Selifonov and Stemmer “Methods ofPopulating Data Structures for Use in Evolutionary Simulations;” WO00/42560 by Selifonov et al., “Methods for Making Character Strings,Polynucleotides & Polypeptides Having Desired Characteristics;”PCT/US00/26708 by Welch et al., “Use of Codon-Varied OligonucleotideSynthesis for Synthetic Shuffling;” and PCT/US01/06775 “Single-StrandedNucleic Acid Template-Mediated Recombination and Nucleic Acid FragmentIsolation” by Affholter.

Several different general classes of sequence modification methods, suchas mutation, recombination, etc. are applicable to the present inventionand set forth, e.g., in the references above and below. The followingexemplify some of the different types of preferred formats for diversitygeneration in the context of the present invention, including, e.g.,certain recombination based diversity generation formats.

Nucleic acids can be recombined in vitro by any of a variety oftechniques discussed in the references above, including e.g., DNAsedigestion of nucleic acids to be recombined followed by ligation and/orPCR reassembly of the nucleic acids. For example, sexual PCR mutagenesiscan be used in which random (or pseudo random, or even non-random)fragmentation of the DNA molecule is followed by recombination, based onsequence similarity, between DNA molecules with different but relatedDNA sequences, in vitro, followed by fixation of the crossover byextension in a polymerase chain reaction. This process and many processvariants is described in several of the references above, e.g., inStemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751.

Similarly, nucleic acids can be recursively recombined in vivo, e.g., byallowing recombination to occur between nucleic acids in cells. Manysuch in vivo recombination formats are set forth in the references notedabove. Such formats optionally provide direct recombination betweennucleic acids of interest, or provide recombination between vectors,viruses, plasmids, etc., comprising the nucleic acids of interest, aswell as other formats. Details regarding such procedures are found inthe references noted above.

Whole genome recombination methods can also be used in which wholegenomes of cells or other organisms are recombined, optionally includingspiking of the genomic recombination mixtures with desired librarycomponents (e.g., genes corresponding to the pathways of the presentinvention). These methods have many applications, including those inwhich the identity of a target gene is not known. Details on suchmethods are found, e.g., in WO 98/31837 by del Cardayre et al.“Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” and in, e.g., PCT/US99/15972 by del Cardayre et al.,also entitled “Evolution of Whole Cells and Organisms by RecursiveSequence Recombination.”

Synthetic recombination methods can also be used, in whicholigonucleotides corresponding to targets of interest are synthesizedand reassembled in PCR or ligation reactions which includeoligonucleotides which correspond to more than one parental nucleicacid, thereby generating new recombined nucleic acids. Oligonucleotidescan be made by standard nucleotide addition methods, or can be made,e.g., by tri-nucleotide synthetic approaches. Details regarding suchapproaches are found in the references noted above, including, e.g., WO00/42561 by Crameri et al., “Olgonucleotide Mediated Nucleic AcidRecombination;” PCT/US00/26708 by Welch et al., “Use of Codon-VariedOligonucleotide Synthesis for Synthetic Shuffling;” WO 00/42560 bySelifonov et al., “Methods for Making Character Strings, Polynucleotidesand Polypeptides Having Desired Characteristics;” and WO 00/42559 bySelifonov and Stemmer “Methods of Populating Data Structures for Use inEvolutionary Simulations.”

In silico methods of recombination can be effected in which geneticalgorithms are used in a computer to recombine sequence strings whichcorrespond to homologous (or even non-homologous) nucleic acids. Theresulting recombined sequence strings are optionally converted intonucleic acids by synthesis of nucleic acids which correspond to therecombined sequences, e.g., in concert with oligonucleotidesynthesis/gene reassembly techniques. This approach can generate random,partially random or designed variants. Many details regarding in silicorecombination, including the use of genetic algorithms, geneticoperators and the like in computer systems, combined with generation ofcorresponding nucleic acids (and/or proteins), as well as combinationsof designed nucleic acids and/or proteins (e.g., based on cross-oversite selection) as well as designed, pseudo-random or randomrecombination methods are described in WO 00/42560 by Selifonov et al.,“Methods for Making Character Strings, Polynucleotides and PolypeptidesHaving Desired Characteristics” and WO 00/42559 by Selifonov and Stemmer“Methods of Populating Data Structures for Use in EvolutionarySimulations.” Extensive details regarding in silico recombinationmethods are found in these applications. This methodology is generallyapplicable to the present invention in providing for recombination ofthe molecules in silico and/or the generation of corresponding nucleicacids or proteins.

Many methods of accessing natural diversity, e.g., by hybridization ofdiverse nucleic acids or nucleic acid fragments to single-strandedtemplates, followed by polymerization and/or ligation to regeneratefull-length sequences, optionally followed by degradation of thetemplates and recovery of the resulting modified nucleic acids can besimilarly used. In one method employing a single-stranded template, thefragment population derived from the genomic library(ies) is annealedwith partial, or, often approximately full length ssDNA or RNAcorresponding to the opposite strand. Assembly of complex chimeric genesfrom this population is then mediated by nuclease-base removal ofnon-hybridizing fragment ends, polymerization to fill gaps between suchfragments and subsequent single stranded ligation. The parentalpolynucleotide strand can be removed by digestion (e.g., if RNA oruracil-containing), magnetic separation under denaturing conditions (iflabeled in a manner conducive to such separation) and other availableseparation/purification methods. Alternatively, the parental strand isoptionally co-purified with the chimeric strands and removed duringsubsequent screening and processing steps. Additional details regardingthis approach are found, e.g., in “Single-Stranded Nucleic AcidTemplate-Mediated Recombination and Nucleic Acid Fragment Isolation” byAffholter, PCT/US01/06775.

In another approach, single-stranded molecules are converted todouble-stranded DNA (dsDNA) and the dsDNA molecules are bound to a solidsupport by ligand-mediated binding. After separation of unbound DNA, theselected DNA molecules are released from the support and introduced intoa suitable host cell to generate a library enriched sequences whichhybridize to the probe. A library produced in this manner provides adesirable substrate for further diversification using any of theprocedures described herein.

Any of the preceding general recombination formats can be practiced in areiterative fashion (e.g., one or more cycles of mutation/recombinationor other diversity generation methods, optionally followed by one ormore selection methods) to generate a more diverse set of recombinantnucleic acids.

Mutagenesis employing polynucleotide chain termination methods have alsobeen proposed (see e.g., U.S. Pat. No. 5,965,408, “Method of DNAreassembly by interrupting synthesis” to Short, and the referencesabove), and can be applied to the present invention. In this approach,double stranded DNAs corresponding to one or more genes sharing regionsof sequence similarity are combined and denatured, in the presence orabsence of primers specific for the gene. The single strandedpolynucleotides are then annealed and incubated in the presence of apolymerase and a chain terminating reagent (e.g., ultraviolet, gamma orX-ray irradiation; ethidium bromide or other intercalators; DNA bindingproteins, such as single strand binding proteins, transcriptionactivating factors, or histones; polycyclic aromatic hydrocarbons;trivalent chromium or a trivalent chromium salt; or abbreviatedpolymerization mediated by rapid thermocycling; and the like), resultingin the production of partial duplex molecules. The partial duplexmolecules, e.g., containing partially extended chains, are thendenatured and reannealed in subsequent rounds of replication or partialreplication resulting in polynucleotides which share varying degrees ofsequence similarity and which are diversified with respect to thestarting population of DNA molecules. Optionally, the products, orpartial pools of the products, can be amplified at one or more stages inthe process. Polynucleotides produced by a chain termination method,such as described above, are suitable substrates for any other describedrecombination format.

Diversity also can be generated in nucleic acids or populations ofnucleic acids using a recombinational procedure termed “incrementaltruncation for the creation of hybrid enzymes” (“ITCHY”) described inOstermeier et al. (1999) “A combinatorial approach to hybrid enzymesindependent of DNA homology” Nature Biotech 17:1205. This approach canbe used to generate an initial a library of variants which canoptionally serve as a substrate for one or more in vitro or in vivorecombination methods. See, also, Ostermeier et al. (1999)“Combinatorial Protein Engineering by Incremental Truncation,” Proc.Natl. Acad. Sci. USA, 96: 3562-67; Ostermeier et al. (1999),“Incremental Truncation as a Strategy in the Engineering of NovelBiocatalysts,” Biological and Medicinal Chemistry, 7: 2139-44.

Mutational methods which result in the alteration of individualnucleotides or groups of contiguous or non-contiguous nucleotides can befavorably employed to introduce nucleotide diversity. Many mutagenesismethods are found in the above-cited references; additional detailsregarding mutagenesis methods can be found in following, which can alsobe applied to the present invention. For example, error-prone PCR can beused to generate nucleic acid variants. Using this technique, PCR isperformed under conditions where the copying fidelity of the DNApolymerase is low, such that a high rate of point mutations is obtainedalong the entire length of the PCR product. Examples of such techniquesare found in the references above and, e.g., in Leung et al. (1989)Technique 1:11-15 and Caldwell et al. (1992) PCR Methods Applic.2:28-33. Similarly, assembly PCR can be used, in a process whichinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions can occur inparallel in the same reaction mixture, with the products of one reactionpriming the products of another reaction.

Oligonucleotide directed mutagenesis can be used to introducesite-specific mutations in a nucleic acid sequence of interest. Examplesof such techniques are found in the references above and, e.g., inReidhaar-Olson et al. (1988) Science, 241:53-57. Similarly, cassettemutagenesis can be used in a process that replaces a small region of adouble stranded DNA molecule with a synthetic oligonucleotide cassettethat differs from the native sequence. The oligonucleotide can contain,e.g., completely and/or partially randomized native sequence(s).

Recursive ensemble mutagenesis is a process in which an algorithm forprotein mutagenesis is used to produce diverse populations ofphenotypically related mutants, members of which differ in amino acidsequence. This method uses a feedback mechanism to monitor successiverounds of combinatorial cassette mutagenesis. Examples of this approachare found in Arkin & Youvan (1992) Proc. Natl. Acad. Sci. USA89:7811-7815. Exponential ensemble mutagenesis can be used forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants. Small groups of residues in a sequence of interestare randomized in parallel to identify, at each altered position, aminoacids which lead to functional proteins. Examples of such procedures arein Delegrave & Youvan (1993) Biotechnology Research 11:1548-1552.

In vivo mutagenesis can be used to generate random mutations in anycloned DNA of interest by propagating the DNA, e.g., in a strain of E.coli that carries mutations in one or more of the DNA repair pathways.These “mutator” strains have a higher random mutation rate than that ofa wild-type parent. Propagating the DNA in one of these strains willeventually generate random mutations within the DNA. Such procedures aredescribed in the references noted above.

Other procedures for introducing diversity into a genome, e.g. abacterial, fungal, animal or plant genome can be used in conjunctionwith the above described and/or referenced methods. For example, inaddition to the methods above, techniques have been proposed whichproduce nucleic acid multimers suitable for transformation into avariety of species (see, e.g., Schellenberger U.S. Pat. No. 5,756,316and the references above). Transformation of a suitable host with suchmultimers, consisting of genes that are divergent with respect to oneanother, (e.g., derived from natural diversity or through application ofsite directed mutagenesis, error prone PCR, passage through mutagenicbacterial strains, and the like), provides a source of nucleic aciddiversity for DNA diversification, e.g., by an in vivo recombinationprocess as indicated above.

Alternatively, a multiplicity of monomeric polynucleotides sharingregions of partial sequence similarity can be transformed into a hostspecies and recombined in vivo by the host cell. Subsequent rounds ofcell division can be used to generate libraries, members of which,include a single, homogenous population, or pool of monomericpolynucleotides. Alternatively, the monomeric nucleic acid can berecovered by standard techniques, e.g., PCR and/or cloning, andrecombined in any of the recombination formats, including recursiverecombination formats, described above.

Methods for generating multispecies expression libraries have beendescribed (in addition to the reference noted above, see, e.g., Petersonet al. (1998) U.S. Pat. No. 5,783,431 “METHODS FOR GENERATING ANDSCREENING NOVEL METABOLIC PATHWAYS,” and Thompson, et al. (1998) U.S.Pat. No. 5,824,485 METHODS FOR GENERATING AND SCREENING NOVEL METABOLICPATHWAYS) and their use to identify protein activities of interest hasbeen proposed (In addition to the references noted above, see Short(1999) U.S. Pat. No. 5,958,672 “PROTEIN ACTIVITY SCREENING OF CLONESHAVING DNA FROM UNCULTIVATED MICROORGANISMS”). Multispecies expressionlibraries include, in general, libraries comprising cDNA or genomicsequences from a plurality of species or strains, operably linked toappropriate regulatory sequences, in an expression cassette. The cDNAand/or genomic sequences are optionally randomly ligated to furtherenhance diversity. The vector can be a shuttle vector suitable fortransformation and expression in more than one species of host organism,e.g., bacterial species, eukaryotic cells. In some cases, the library isbiased by preselecting sequences which encode a protein of interest, orwhich hybridize to a nucleic acid of interest. Any such libraries can beprovided as substrates for any of the methods herein described.

The above-described procedures have been largely directed to increasingnucleic acid and/or encoded protein diversity. However, in many cases,not all of the diversity is useful, e.g., functional, and contributesmerely to increasing the background of variants that must be screened orselected to identify the few favorable variants. In some applications,it is desirable to preselect or prescreen libraries (e.g., an amplifiedlibrary, a genomic library, a cDNA library, a normalized library, etc.)or other substrate nucleic acids prior to diversification, e.g., byrecombination-based mutagenesis procedures, or to otherwise bias thesubstrates towards nucleic acids that encode functional products. Forexample, in the case of antibody engineering, it is possible to bias thediversity generating process toward antibodies with functional antigenbinding sites by taking advantage of in vivo recombination events priorto manipulation by any of the described methods. For example, recombinedCDRs derived from B cell cDNA libraries can be amplified and assembledinto framework regions (e.g., Jirholt et al. (1998) “Exploiting sequencespace: shuffling in vivo formed complementarity determining regions intoa master framework” Gene 215: 471) prior to diversifying according toany of the methods described herein.

Libraries can be biased towards nucleic acids which encode proteins withdesirable enzyme activities. For example, after identifying a clone froma library which exhibits a specified activity, the clone can bemutagenized using any known method for introducing DNA alterations. Alibrary comprising the mutagenized homologues is then screened for adesired activity, which can be the same as or different from theinitially specified activity. An example of such a procedure is proposedin Short (1999) U.S. Pat. No. 5,939,250 for “PRODUCTION OF ENZYMESHAVING DESIRED ACTIVITIES BY MUTAGENESIS.” Desired activities can beidentified by any method known in the art. For example, WO 99/10539proposes that gene libraries can be screened by combining extracts fromthe gene library with components obtained from metabolically rich cellsand identifying combinations which exhibit the desired activity. It hasalso been proposed (e.g., WO 98/58085) that clones with desiredactivities can be identified by inserting bio active substrates intosamples of the library, and detecting bio active fluorescencecorresponding to the product of a desired activity as described hereinusing a fluorescent analyzer, e.g., a flow cytometry device, a CCD, afluorometer, or a spectrophotometer.

Libraries can also be biased towards nucleic acids which have specifiedcharacteristics, e.g., hybridization to a selected nucleic acid probe.For example, application WO 99/10539 proposes that polynucleotidesencoding a desired activity (e.g., an enzymatic activity, for example: alipase, an esterase, a protease, a glycosidase, a glycosyl transferase,a phosphatase, a kinase, an oxygenase, a peroxidase, a hydrolase, ahydratase, a nitrilase, a transaminase, an amidase or an acylase) can beidentified from among genomic DNA sequences in the following manner.Single stranded DNA molecules from a population of genomic DNA arehybridized to a ligand-conjugated probe. The genomic DNA can be derivedfrom either a cultivated or uncultivated microorganism, or from anenvironmental sample. Alternatively, the genomic DNA can be derived froma multicellular organism, or a tissue derived therefrom. Second strandsynthesis can be conducted directly from the hybridization probe used inthe capture, with or without prior release from the capture medium or bya wide variety of other strategies known in the art. Alternatively, theisolated single-stranded genomic DNA population can be fragmentedwithout further cloning and used directly in, e.g., arecombination-based approach, that employs a single-stranded template,as described above.

“Non-Stochastic” methods of generating nucleic acids and polypeptidesare alleged in Short “Non-Stochastic Generation of Genetic Vaccines andEnzymes” WO 00/46344. These methods, including proposed non-stochasticpolynucleotide reassembly and site-saturation mutagenesis methods beapplied to the present invention as well. Random or semi-randommutagenesis using doped or degenerate oligonucleotides is also describedin, e.g., Arkin and Youvan (1992) “Optimizing nucleotide mixtures toencode specific subsets of amino acids for semi-random mutagenesis”Biotechnology 10:297-300; Reidhaar-Olson et al. (1991) “Randommutagenesis of protein sequences using oligonucleotide cassettes”Methods Enzymol. 208:564-86; Lim and Sauer (1991) “The role of internalpacking interactions in determining the structure and stability of aprotein” J. Mol. Biol. 219:359-76; Breyer and Sauer (1989) “Mutationalanalysis of the fine specificity of binding of monoclonal antibody 51Fto lambda repressor” J. Biol. Chem. 264:13355-60); and “Walk-ThroughMutagenesis” (Crea, R; U.S. Pat. Nos. 5,830,650 and 5,798,208, and EPPatent 0527809 B1.

It will readily be appreciated that any of the above describedtechniques suitable for enriching a library prior to diversification canalso be used to screen the products, or libraries of products, producedby the diversity generating methods.

Kits for mutagenesis, library construction and other diversitygeneration methods are also commercially available. For example, kitsare available from, e.g., Stratagene (e.g., QuickChange™ site-directedmutagenesis kit; and Chameleon™ double-stranded, site-directedmutagenesis kit), Bio/Can Scientific, Bio-Rad (e.g., using the Kunkelmethod described above), Boehringer Mannheim Corp., ClonetechLaboratories, DNA Technologies, Epicentre Technologies (e.g., 5 prime 3prime kit); Genpak Inc, Lemargo Inc, Life Technologies (Gibco BRL), NewEngland Biolabs, Pharmacia Biotech, Promega Corp., QuantumBiotechnologies, Amersham International plc (e.g., using the Ecksteinmethod above), and Anglian Biotechnology Ltd (e.g., using theCarter/Winter method above).

The above references provide many mutational formats, includingrecombination, recursive recombination, recursive mutation andcombinations or recombination with other forms of mutagenesis, as wellas many modifications of these formats. Regardless of the diversitygeneration format that is used, the nucleic acids of the invention canbe recombined (with each other, or with related (or even unrelated)sequences) to produce a diverse set of recombinant nucleic acids,including, e.g., sets of homologous nucleic acids, as well ascorresponding polypeptides.

A recombinant nucleic acid produced by recombining one or morepolynucleotide sequences of the invention with one or more additionalnucleic acids using any of the above-described formats alone or incombination also forms a part of the invention. The one or moreadditional nucleic acids may include another polynucleotide of theinvention; optionally, alternatively, or in addition, the one or moreadditional nucleic acid can include, e.g., a nucleic acid encoding anaturally-occurring interferon-alpha or a subsequence thereof, or anyhomologous interferon-alpha or subsequence thereof (e.g., as found inGenBank or other available literature), or, e.g., any other homologousor non-homologous nucleic acid or fragments thereof (certainrecombination formats noted above, notably those performed syntheticallyor in silico, do not require homology for recombination).

Therapeutic Uses

Various interferon-alpha polypeptides and interferon-alpha conjugateshave been approved or are in clinical development for treatment of avariety of diseases or conditions such as Chronic Hepatitis C, ChronicHepatitis B, Hairy Cell Leukemia, Malignant Melanoma, FollicularLymphoma, Condylomata Acuminata, AIDS-related Kaposi's Sarcoma,Non-Hodgkin's Lymphoma, Chronic Melogenous Leukemia, Basal CellCarcinoma, Multiple Myeloma, carcinoid tumors, bladder cancer, Crohn'sdisease, Cutaneous T Cell Lymphoma, Renal Cell Carcinoma, MultipleSclerosis, and AIDS. Accordingly, the present invention contemplates theuse of a composition comprising one or more polypeptide or conjugate ofthe invention (i.e., a “composition of the invention”) to treat adisease or condition which is responsive to an interferon-alphapolypeptide and/or an interferon-alpha conjugate, such as a conditiondescribed above, or any other disease or condition which is responsiveto a polypeptide or a conjugate of the invention.

Treatment of Viral Infections and Conditions Associated with ViralInfection

In one aspect, the invention provides a method for treating a subjectinfected with a virus, comprising administering to the subject acomposition of the invention in an amount effective to decrease thelevel of the virus in the subject and/or to ameliorate a symptom orcondition associated with the viral infection. Exemplary viralinfections contemplated for treatment methods of the invention include,but are not limited to, infection by a virus of the Flaviviridae family,such as, for example, Hepatitis C Virus, Yellow Fever Virus, West NileVirus, Japanese Encephalitis Virus, Dengue Virus, and Bovine ViralDiarrhea Virus; infection by a virus of the Hepadnaviridae family, suchas, for example, Hepatitis B Virus; infection by a virus of thePicornaviridae family, such as, for example, Encephalomyocarditis Virus,Human Rhinovirus, and Hepatitis A Virus; infection by a virus of theRetroviridae family, such as, for example, Human Immunodeficiency Virus,Simian Immunodeficiency Virus, Human T-Lymphotropic Virus, and RousSarcoma Virus; infection by a virus of the Coronaviridae family, suchas, for example, SARS coronavirus; infection by a virus of theRhabdoviridae family, such as, for example, Rabies Virus and VesicularStomatitis Virus; infection by a virus of the Paramyxoviridae family,such as, for example, Respiratory Syncytial Virus and ParainfluenzaVirus; infection by a virus of the Papillomaviridae family, such as, forexample, Human Papillomavirus; and infection by a virus of theHerpesviridae family, such as, for example, Herpes Simplex Virus.

The following provides non-limiting examples for treatment of exemplaryviral infections and diseases and conditions associated with suchinfections, using polypeptides and conjugates of the invention,including suggested dosing schedules for polypeptides and conjugates ofthe invention and approaches to monitoring the efficacy of suchtreatments. The dosing schedules of polypeptides or conjugates of theinvention for the treatment of other viral infections and diseases andconditions associated with viral infections, and approaches tomonitoring the efficacy of such treatments, is ascertainable by oneskilled in the art.

Hepatitis C Virus

In one aspect the invention provides a method of treating a patientinfected with Hepatitis C Virus (HCV), comprising administering to thepatient an effective amount of a composition of the invention comprisingone or more polypeptide or conjugate of the invention. The inventionalso provides a composition for use in treating a patient infected withHCV, comprising one or more polypeptide or conjugate of the inventionand a pharmaceutically acceptable carrier or excipient. A patientdiagnosed as infected with HCV includes a patient exhibiting HCV RNA inthe blood and/or exhibiting anti-HCV antibody in the serum.

A composition comprising a polypeptide of the invention will generallybe administered at a dose and frequency similar to what is employed inHCV therapeutic regimens using clinically-approved interferon-alphapolypeptides, such as, e.g. ROFERON®-A (Interferon alfa-2a, recombinant;Hoffmann-La Roche Inc.), INTRON® A (Interferon alfa-2b, recombinant;Schering Corporation), and INFERGEN® (interferon alfacon-1; InterMune,Inc.). Exemplary recommended dosing schedules of ROFERON or INTRON A forthe treatment of chronic HCV is 3 million IU (approximately 15micrograms (mcg)) three times a week by subcutaneous injection for,e.g., 24 to 48 weeks. An exemplary recommended dosing schedule ofINFERGEN for the treatment of chronic HCV is 9 mcg three times a week bysubcutaneous injection for, e.g., 24 to 48 weeks. Depending on a numberof factors (including but not limited to the activity and thepharmacokinetics of the polypeptide of the invention and the size andhealth of the patient), the polypeptide may be administered in loweramounts (such as, for example, about 2, 3, 4, 5, 6, 7, or 8 mcg) and/orless frequently (such as once per week or twice per week) than describedabove.

Likewise, a composition comprising a conjugate of the invention willgenerally be administered at a dose and frequency similar to what isemployed in HCV therapeutic regimens using clinically-approvedinterferon-alpha conjugates, such as, e.g., PEGASYS® (Peginterferonalfa-2a; Hoffmann-La Roche, Inc.) or PEG-INTRON® (peginterferon alfa-2b;Schering Corporation). An exemplary recommended dosing schedule ofPEGASYS for the treatment of chronic HCV is 180 mcg once weekly bysubcutaneous injection for, e.g., 24 to 48 weeks. Depending on a numberof factors (including but not limited to the molecular weight, activity,and pharmacokinetics of the conjugate of the invention and the size andhealth of the patient), the conjugate may be administered in loweramounts (such as, for example, about 25, 50, 75, 100, 125, or 150 mcg)and/or less frequently (such as once every 10 days, or once every 2weeks) than described above.

In some instances the polypeptide or conjugate of the invention isadministered in combination with one or more additional therapeuticagent(s). For example, the polypeptide or conjugate of the invention maybe administered in combination with a small-molecule antiviral drug suchas Ribavirin, which is sold under the names COPEGUS® (Hoffmann-La Roche,Inc) and REBETOL® (Schering Corporation). Alternatively, or in additionto a small-molecule antiviral drug, the polypeptide or conjugate of theinvention may be administered in combination with one or more additionalcytokine, such as, for example, IFN-gamma, which is sold under the nameActimmune® (interferon gamma-1b; InterMune, Inc.), IL-2, which is soldunder the name PROLEUKIN® IL-2 (aldesleukin recombinant humaninterleukin-2 (rhIL-2); Chiron Corp.), or IL-12 (interleukin-12).

The precise amount and frequency of administration of the polypeptide orconjugate of the invention will depend on a number of factors such asthe specific activity and the pharmacokinetic properties of thepolypeptide or the conjugate, as well as the nature of the conditionbeing treated (such as, the genotype of the Hepatitis C virus beingtreated), among other factors known to those of skill in the art.Normally, the dose should be capable of preventing or lessening theseverity or spread of the indication being treated. Such a dose may betermed an “effective” or “therapeutically effective” amount. It will beapparent to those of skill in the art that an effective amount of apolypeptide, conjugate or composition of the invention depends, interalia, upon the condition being treated, the dose, the administrationschedule, whether the polypeptide or conjugate or composition isadministered alone or in combination with other therapeutic agents, theserum half-life and other pharmacokinetic properties of the polypeptide,conjugate or composition, as well as the size, age, and general healthof the patient. The dosage and frequency of administration isascertainable by one skilled in the art using known techniques.

The effectiveness of treatment may be determined by measuring viralload, for example by determining the titer or level of virus in serum orplasma using methods known in the art, such as, e.g., by monitoringviral RNA levels using quantitative PCR-based tests, such as the COBASAMPLICOR® HCV Test, v2.0 or the COBAS AMPLICOR HCV MONITOR® Test, v2.0(both from Roche Diagnostics). In some instances, an effective amount ofa composition of the invention is one that is sufficient to achieve areduction in viral load by at least 2 log units, at least 3 log units,at least 4 log units, at least 5 log units, at least 6 log units or atleast 7 log units over the course of treatment, compared to the viralload prior to treatment (which is generally in the range of 10⁵-10⁷copies of HCV RNA/ml for chronic HCV patients). In some instances aneffective amount of a composition of the invention is an amount that issufficient to reduce viral load to levels which are essentiallyundetectable, such as, for example, less than about 500 copies/ml serumor less than about 100 copies/ml serum. The invention includes a methodof reducing the level of HCV RNA in serum of a patient infected withHCV, comprising administering to the patient a composition of theinvention in an amount effective to reduce the level of HCV RNA comparedto the HCV RNA level present prior to the start of treatment.

The effectiveness of treatment may alternatively or in addition bedetermined by measuring a parameter indicative of a condition associatedwith HCV infection, such as, e.g., liver damage. For example, the levelof serum alanine aminotransferase (ALT) may be measured using a standardassay. In general, an ALT level of less than about 50 internationalunits/ml (IU/ml) serum is considered normal. A higher ALT level may beindicative of ongoing liver damage. In some instances, an effectiveamount of a composition of the invention is an amount effective toreduce ALT level, in a patient with a higher than normal ALT level, toless than about 50 IU/ml of serum. Thus, the invention includes a methodof reducing the serum ALT level of a patient infected with HCVexhibiting an initial ALT level greater than 50 IU/ml, comprisingadministering to the patient a composition of the invention in an amounteffective to reduce the ALT level to less than about 50 IU/ml.

Human Immunodeficiency Virus

In another aspect the invention provides a method of treating a patientinfected with Human Immunodeficiency Virus (HIV), such as HIV-1 orHIV-2, or a disease or condition associated with HIV infection, such as,for example, AIDS-related Kaposi's sarcoma, comprising administering tothe patient an effective amount of a composition of the inventioncomprising one or more polypeptide or conjugate of the invention,optionally in association with other antiviral therapeutic agents asdescribed below. The invention also provides a composition for use intreating a patient infected with HIV or a disease or conditionassociated with HIV infection, comprising one or more polypeptide orconjugate of the invention and a pharmaceutically acceptable carrier orexcipient. A patient diagnosed as infected with HIV includes a patientexhibiting detectable levels of HIV RNA or proviral DNA in the blood,and/or exhibiting detectable levels of p24 antigen or anti-HIV antibodyin serum.

A composition comprising a polypeptide of the invention will generallybe administered at a dose and frequency similar to what is employed inHIV therapeutic regimens using interferon-alpha polypeptides such as,e.g. ROFERON®-A (Interferon alfa-2a, recombinant; Hoffmann-La RocheInc.), INTRON® A (Interferon alfa-2b, recombinant; ScheringCorporation), and INFERGEN® (interferon alfacon-1; InterMune, Inc.). Aswas noted above, exemplary recommended dosing schedules of ROFERON orINTRON A for the treatment of chronic HCV is 3 million IU (approximately15 micrograms (mcg)) three times a week by subcutaneous injection for,e.g., 24 to 48 weeks, and a exemplary recommended dosing schedule ofINFERGEN for the treatment of chronic HCV is 9 mcg three times a week bysubcutaneous injection for, e.g., 24 to 48 weeks. An exemplaryrecommended dosing schedule of ROFERON for the treatment of AIDS-relatedKaposi's sarcoma is 36 million units daily for 10 to 12 weeks, then 36million units 3 times a week. An exemplary recommended dosing scheduleof INTRON A for the treatment of AIDS-related Kaposi's sarcoma is 30million IU/m2 three times a week administered subcutaneously. Suchdosing schedules provide useful ranges for dosage of a polypeptide ofthe invention for the treatment of HIV or a disease or conditionassociated with HIV infection. Depending on a number of factors(including but not limited to the activity and the pharmacokinetics ofthe polypeptide of the invention and the size, age and health of thepatient), the polypeptide of the invention may be administered in loweramounts and/or less frequently than described above.

Likewise, a composition comprising a conjugate of the invention willgenerally be administered at a dose and frequency similar to what isemployed in HIV therapeutic regimens using interferon-alpha conjugates,such as, e.g., PEGASYS® (Peginterferon alfa-2a; Hoffmann-La Roche, Inc.)or PEG-INTRON® (peginterferon alfa-2b; Schering Corporation). Anexemplary dosing schedule of PEG-INTRON for the treatment of HIV isbetween about 1.0 mcg/kg/week and 3.0 mcg/kg/week by subcutaneousinjection for, e.g., 24 to 48 weeks. Such a dosing schedule provides auseful range for dosage of a conjugate of the invention for thetreatment of HIV. Depending on a number of factors (including but notlimited to the molecular weight, activity, and pharmacokinetics of theconjugate of the invention and the size, age and health of the patient),the conjugate may be administered in lower amounts (such as, forexample, about 0.1, 0.25, 0.50, or 0.75 mcg/kg/week) and/or lessfrequently (such as once every 10 days, or once every 2 weeks) thandescribed above.

In some instances the polypeptide or conjugate of the invention isadministered in combination with one or more additional therapeuticagent(s). Current clinical treatments of HIV-1 infection in man includemulti-drug combination therapies generally termed Highly ActiveAntiretroviral Therapy (“HAART”). The polypeptide or conjugate of theinvention may thus be administered in combination with HAART or otherantiviral therapeutic compounds. Typical components of HAART, whichinvolve various combinations of nucleoside reverse transcriptaseinhibitors (“NRTI”), non-nucleoside reverse transcriptase inhibitors(“NNRTI”) and HIV protease inhibitors (“PI”), are described, forexample, in A. M. Vandamme et al. (1998) Antiviral Chemistry &Chemotherapy, 9:187-203; “Drugs for HIV Infection” in The Medical LetterVol. 39 (Issue 1015) Dec. 5, 1997, pages 111-116; and published UnitedStates patent application US 20020182179 A1; each of which isincorporated by reference herein. If the HIV-infected patient is alsoinfected with HCV, the polypeptide or conjugate of the invention may beadministered in combination with an antiviral drug such as Ribavirin,which is sold under the names COPEGUS® (Hoffmann-La Roche, Inc) andREBETOL® (Schering Corporation), along with HAART.

The precise amount and frequency of administration of the polypeptide orconjugate of the invention, and administration of additional therapeuticagents such as HAART and/or Ribavirin, will depend on a number offactors such as the specific activity and the pharmacokinetic propertiesof the polypeptide or the conjugate, as well as the nature of thecondition being treated (such as, the presence of additional viralinfections such as HCV), among other factors known to those of skill inthe art. Normally, the dose should be capable of preventing or lesseningthe severity or spread of the indication being treated. Such a dose maybe termed an “effective” or “therapeutically effective” amount. It willbe apparent to those of skill in the art that an effective amount of apolypeptide, conjugate or composition of the invention depends, interalia, upon the condition being treated, the dose, the administrationschedule, whether the polypeptide or conjugate or composition isadministered alone or in combination with other therapeutic agents, theserum half-life and other pharmacokinetic properties of the polypeptide,conjugate or composition, as well as the size, age, and general healthof the patient. The dosage and frequency of administration isascertainable by one skilled in the art using known techniques.

In addition to general uses described above, a polypeptide or conjugateof the invention may be administered to the following subsets ofpatients infected with HIV: as an adjuvant therapy, for example to HAARTas described above; as monotherapy or combination therapy in early stagepatients when the viral load is generally high; as a combined anti-viraland immunodulatory agent for patients undergoing structured treatmentinterruptions (STI) or “drug holidays”; as salvage therapy in patientswhose HAART options are limited; as an antiviral method of treatment tokeep viral load in check without initiating HAART therapy in order todelay the appearance of HAART resistant virus.

The effectiveness of treatment may be determined by measuring viralload, for example by determining the titer or level of virus in serum orplasma using methods known in the art, such as, e.g., by monitoringHIV-1 viral RNA levels using quantitative RT-PCR based tests, such asthe AMPLICOR HIV-1 MONITOR® Test, v1.5 (Roche Diagnostics). In someinstances, an effective amount of a composition of the invention is onethat is sufficient to achieve a reduction in viral load by at least 0.5log units, at least 1 log unit, at least 2 log units, at least 3 logunits, at least 4 log units, at least 5 log units, at least 6 log unitsor at least 7 log units over the course of treatment, compared to theviral load prior to treatment. In some instances an effective amount ofa composition of the invention is an amount that is sufficient to reduceviral load to levels which are essentially undetectable, such as, forexample, less than about 50-100 copies HIV-1 RNA per ml serum. Theinvention includes a method of reducing the level of HIV RNA in serum ofa patient infected with HIV, comprising administering to the patient acomposition of the invention in an amount effective to reduce the levelof HIV RNA compared to the HIV RNA level present prior to the start oftreatment.

The effectiveness of treatment may alternatively or in addition bedetermined by a serum markers for HIV replication, such as the presenceof HIV p24 antigen in the blood. In some instances an effective amountof a composition of the invention is an amount that is sufficient toreduce the level of p24 antigen in the blood to 50%, 25%, 10% or 5% ofthe level present prior to the start of treatment. In some instances aneffective amount of a composition of the invention is an amount that issufficient to reduce the level of p24 antigen to a level which isessentially undetectable. The invention includes a method of reducingthe level of p24 antigen in serum of a patient infected with HIV,comprising administering to the patient a composition of the inventionin an amount effective to reduce the level of p24 antigen compared tothe p24 antigen level present prior to the start of treatment.

Hepatitis B Virus

In another aspect, the invention provides a method of treating a patientinfected with Hepatitis B Virus (HBV), comprising administering to thepatient an effective amount of a composition of the invention comprisingone or more polypeptide or conjugate of the invention. The inventionalso provides a composition for use in treating a patient infected withHBV, comprising one or more polypeptide or conjugate of the inventionand a pharmaceutically acceptable carrier or excipient.

A patient diagnosed as infected with HBV exhibits detectable hepatitis Bsurface antigen (HBsAg) in the serum. Chronic HBV infection is furthercategorized as either “replicative” or “non-replicative”. In replicativeinfection, the patient usually has a relatively high serum concentrationof viral DNA and detectable HBeAg, which is an alternatively processedprotein of the HBV pre-core gene that is synthesized under conditions ofhigh viral replication. However, in rare strains of HBV with mutationsin the pre-core gene, replicative infection can occur in the absence ofdetectable serum HBeAg. Patients with chronic hepatitis B andreplicative infection have a generally worse prognosis and a greaterchance of developing cirrhosis and/or hepatocellular carcinoma thanthose without HBeAg. In non-replicative infection, the rate of viralreplication in the liver is low, serum HBV DNA concentration isgenerally low and hepatitis Be antigen (HBeAg) is not detected.

A composition comprising a polypeptide of the invention will generallybe administered at a dose and frequency similar to what is employed inHBV therapeutic regimens using clinically-approved interferon-alphapolypeptides, such as, e.g. INTRON® A (Interferon alfa-2b, recombinant;Schering Corporation). An exemplary recommended dosing schedule ofINTRON A for the treatment of chronic HBV in adults is 30 to 35 millionIU per week by subcutaneous or intramuscular injection, either as 5million IU per day (qd) or as 10 million IU three times per week (tiw)for 16 weeks. Depending on a number of factors (including, but notlimited to, the activity and the pharmacokinetics of the polypeptide ofthe invention, and the size and health of the patient), the polypeptideof the invention may be administered in lower amounts (such as, forexample, about 5, 10, 15, 20, or 25 million IU per week) and/or lessfrequently (such as once per week or twice per week) than describedabove.

Likewise, a composition comprising a conjugate of the invention willgenerally be administered at a dose and frequency similar to what isemployed in HBV therapeutic regimens using interferon-alpha conjugatescurrently undergoing clinical trials, such as, e.g., PEGASYS®(Peginterferon alfa-2a; Hoffmann-La Roche, Inc.). Exemplary dosingschedules of PEGASYS for the treatment of chronic HBV is between 90mcg-270 mcg injected once per week for a total of 24 weeks. Depending ona number of factors (including but not limited to the molecular weight,activity, and pharmacokinetics of the conjugate of the invention and thesize and health of the patient), the conjugate may be administered inlower amounts (such as, for example, about 25, 50, 75, 100, 125, 150, or200 mcg) and/or less frequently (such as once every 10 days, or onceevery 2 weeks) than described above.

In some instances the polypeptide or conjugate of the invention isadministered in combination with one or more additional therapeuticagent(s). For example, the polypeptide or conjugate of the invention maybe administered in combination with antiviral drugs such as lamivudine(also known as 3TC), which is sold under the name Epivir-HBV®(GlaxoSmithKline), or adefovir dipivoxil, which is sold under the nameHepsera® (Gilead Sciences).

The precise amount and frequency of administration of the polypeptide orconjugate of the invention will depend on a number of factors such asthe specific activity and the pharmacokinetic properties of thepolypeptide or the conjugate, as well as the nature of the conditionbeing treated (such as, e.g., in the case of chronic HBV infection,whether the infection is replicative or non-replicative), among otherfactors known to those of skill in the art. Normally, the dose should becapable of preventing or lessening the severity or spread of theindication being treated. Such a dose may be termed an “effective” or“therapeutically effective” amount. It will be apparent to those ofskill in the art that an effective amount of a polypeptide, conjugate orcomposition of the invention depends, inter alia, upon the conditionbeing treated, the dose, the administration schedule, whether thepolypeptide or conjugate or composition is administered alone or incombination with other therapeutic agents, the serum half-life and otherpharmacokinetic properties of the polypeptide, conjugate or composition,as well as the size, age, and general health of the patient. The dosageand frequency of administration is ascertainable by one skilled in theart using known techniques.

The effectiveness of treatment may be determined for example bymeasuring the viral load, e.g. the level of viral DNA in serum orplasma, using methods known in the art. Methods for monitoring HBV DNAlevels include quantitative PCR-based tests, such as the COBAS AMPLICORHBV MONITOR® Test, v2.0 or the AMPLICOR HBV MONITOR® Test, v2.0 (bothfrom Roche Diagnostics). In some instances an effective amount of acomposition of the invention is an amount that is sufficient to reduceviral DNA to, e.g., less than about 500,000 copies/ml serum or less thanabout 100,000 copies/ml serum or less than about 10,000 copies/ml serum,or to levels which are essentially undetectable (such as, for example,less than about 1000 copies/ml serum, less than about 500 copies/mlserum, or less than about 200 copies/ml serum). The invention includes amethod of reducing the level of HBV DNA in serum of a patient infectedwith HBV, comprising administering to the patient a composition of theinvention in an amount effective to reduce the level of HBV DNA comparedto the HBV DNA level present prior to the start of treatment.

The effectiveness of treatment may alternatively or in addition bedetermined by measuring other serum markers for HBV replication, such asHBeAg. In some instances an effective amount of a composition of theinvention is an amount that is sufficient to reduce the level of HBeAgin serum to 50%, 25%, 10% or 5% of the level present prior to the startof treatment. In some instances an effective amount of a composition ofthe invention is an amount that is sufficient to reduce the level ofHBeAg to a level which is essentially undetectable. The inventionincludes a method of reducing the level of HBeAg in serum of a patientinfected with HBV, comprising administering to the patient a compositionof the invention in an amount effective to reduce the level of HBeAgcompared to the HBeAg level present prior to the start of treatment.

As discussed above, another serum marker indicative of HBV infection isHBsAg. Thus, the effectiveness of treatment may alternatively or inaddition be determined by measuring the level of HBsAg in the serum. Insome instances an effective amount of a composition of the invention isan amount that is sufficient to reduce the level of HBsAg in serum to50%, 25%, 10% or 5% of the level present prior to the start oftreatment. In some instances an effective amount of a composition of theinvention is an amount that is sufficient to reduce level of HBsAg to alevel which is essentially undetectable. The invention includes a methodof reducing the level of HBsAg in serum of a patient infected with HBV,comprising administering to the patient a composition of the inventionin an amount effective to reduce the level of HBsAg compared to theHBsAg level present prior to the start of treatment.

The effectiveness of treatment may alternatively or in addition bedetermined by measuring a parameter indicative of a condition associatedwith HBV infection, such as, e.g., liver damage. For example, the levelof serum alanine aminotransferase (ALT) may be measured using a standardassay. In general, an ALT level of less than about 50 internationalunits/ml (IU/ml) serum is considered normal. A higher ALT level may beindicative of ongoing liver damage. In some instances, an effectiveamount of a composition of the invention is an amount effective toreduce ALT level, in a patient with a higher than normal ALT level, toless than about 50 IU/ml of serum. Thus, the invention includes a methodof reducing the serum ALT level of a patient infected with HBVexhibiting an initial ALT level greater than 50 IU/ml, comprisingadministering to the patient a composition of the invention in an amounteffective to reduce the ALT level to less than about 50 IU/ml.

Human T-Lymphotropic Virus

In another aspect the invention provides a method of treating a patientinfected with a Human T-Lymphotropic Virus, such as Human T-LymphotropicVirus type 1 (HTLV-1), or a disease or condition associated with HTLV-1infection, such as, for example, adult T-cell leukemia/lymphoma (ATLL),HTLV-1-associated myelopathy (HAM), Tropical Spastic Paraparesis (TSP),uveitis, or arthropathy. The method comprises administering to thepatient an effective amount of a composition of the invention comprisingone or more polypeptide or conjugate of the invention. The inventionalso provides a composition for use in treating a patient infected withHTLV-1, or a disease or condition associated with HTLV-1 infection, thecomposition comprising one or more polypeptide or conjugate of theinvention and a pharmaceutically acceptable carrier or excipient. Apatient diagnosed with HTLV-1 infection includes a patient exhibitingHTLV-1 proviral DNA in the blood and/or antibody to an HTLV-1 antigen inthe serum.

A composition comprising a polypeptide of the invention will generallybe administered at a dose and frequency similar to what is employed inHCV or oncology therapeutic regimens using clinically-approvedinterferon-alpha polypeptides, such as, e.g. ROFERON®-A (Interferonalfa-2a, recombinant; Hoffmann-La Roche Inc.) and INTRON® A (Interferonalfa-2b, recombinant; Schering Corporation). Exemplary recommendeddosing schedules of ROFERON or INTRON A for the treatment of chronic HCVis 3 million IU (approximately 15 micrograms (mcg)) three times a weekby subcutaneous injection for, e.g., 24 to 48 weeks. An exemplaryrecommended dosing schedule of ROFERON for the treatment of hairy-cellleukemia is 3-5 million units daily by subcutaneous injection for 16 to24 weeks, then 3 million units 3 times a week for maintenance. Anexemplary recommended dosing schedule of INTRON A for the treatment ofhairy-cell leukemia is 2 million IU/m2 (square meter of body surface)administered subcutaneously 3 times a week for 6 months. Such dosingschedules provide useful ranges for dosage of a polypeptide of theinvention for the treatment of HTLV-1 infection, or a disease orcondition associated with HTLV-1 infection such as adult T-cellleukemia/lymphoma (ATLL), HTLV-1-associated myelopathy (HAM), orTropical Spastic Paraparesis (TSP). Depending on a number of factors(including but not limited to the activity and the pharmacokinetics ofthe polypeptide of the invention and the size, age and health of thepatient), the polypeptide may be administered in lower amounts and/orless frequently than described above.

Likewise, a composition comprising a conjugate of the invention willgenerally be administered at a dose and frequency similar to what isemployed in HCV therapeutic or oncology therapeutic regimens usingclinically-approved interferon-alpha conjugates, such as, e.g., PEGASYS®(Peginterferon alfa-2a; Hoffmann-La Roche, Inc.) or PEG-INTRON®(peginterferon alfa-2b; Schering Corporation). An exemplary recommendeddosing schedule of PEGASYS for the treatment of chronic HCV is 180 mcgonce weekly by subcutaneous injection for, e.g., 24 to 48 weeks. Anexemplary recommended dosing schedule of PEG-INTRON for the treatment ofchronic myelogenous leukemia is 6 mcg/kg body weight once weekly bysubcutaneous injection for, e.g., 52 weeks. Such dosing schedulesprovide useful ranges for dosage of a conjugate of the invention for thetreatment of HTLV-1 infection, or a disease or condition associated withHTLV-1 infection such as adult T-cell leukemia/lymphoma (ATLL),HTLV-1-associated myelopathy (HAM), or Tropical Spastic Paraparesis(TSP). Depending on a number of factors (including but not limited tothe molecular weight, activity, and pharmacokinetics of the conjugate ofthe invention and the size, age and health of the patient), theconjugate may be administered in lower amounts and/or less frequentlythan described above.

In some instances the polypeptide or conjugate of the invention isadministered in combination with one or more additional therapeuticagent(s). For example, the polypeptide or conjugate of the invention maybe administered in combination with an antiretroviral drug such aszidovudine (AZT) and/or lamivudine (3TC). It may also be administered incombination with peripheral blood stem cell transplantation,conventional chemotherapy, or high dose chemotherapy with autologous orallogeneic bone marrow transplantation. Alternatively, the polypeptideor conjugate of the invention may be combined with other immunotherapy,for example with anti-interleukin-2 receptor monoclonal antibodies orinjection of cytotoxic T-cells directed against virus antigens.

The precise amount and frequency of administration of the polypeptide orconjugate of the invention will depend on a number of factors such asthe specific activity and the pharmacokinetic properties of thepolypeptide or the conjugate, as well as the nature of the conditionbeing treated, among other factors known to those of skill in the art.Normally, the dose should be capable of preventing or lessening theseverity or spread of the indication being treated. Such a dose may betermed an “effective” or “therapeutically effective” amount. It will beapparent to those of skill in the art that an effective amount of apolypeptide, conjugate or composition of the invention depends, interalia, upon the condition being treated, the dose, the administrationschedule, whether the polypeptide or conjugate or composition isadministered alone or in combination with other therapeutic agents, theserum half-life and other pharmacokinetic properties of the polypeptide,conjugate or composition, as well as the size, age, and general healthof the patient. The dosage and frequency of administration isascertainable by one skilled in the art using known techniques.

The effectiveness of treatment may be determined by measuring the HTLV-1viral load, such as, for example, measuring the level of HTLV-1 proviralDNA in the blood using methods known in the art, for example byquantitative PCR as described by Saito et al., (2004) J. Infect Dis.189(1):29-40. In some instances, an effective amount of a composition ofthe invention is one that is sufficient to achieve a reduction in viralload by at least 0.5 log unit, such as at least 1 log unit, at least 2log units, at least 3 log units, at least 4 log units, at least 5 logunits, at least 6 log units, or at least 7 log units over the course oftreatment, compared to the viral load prior to treatment. In someinstances an effective amount of a composition of the invention is anamount that is sufficient to reduce viral load to levels which areessentially undetectable. The invention includes a method of reducingthe level of HTLV-1 proviral DNA in blood of a patient infected withHTLV-1, comprising administering to the patient a composition of theinvention in an amount effective to reduce the level of HTLV-1 proviralDNA compared to that present prior to the start of treatment.

The effectiveness of treatment may alternatively or in addition bedetermined by measuring titer of an anti-HTLV-1 antibody in the serum,using methods known in the art, such as, for example, bycommercially-available tests such as INNO-LIA™ HTLV I/II (Innogenetics;Gent Belgium) and Abbott HTLV-I/HTLV-II EIA (Abbott Laboratories; AbbottPark, Ill.). In some instances an effective amount of a composition ofthe invention is an amount that is sufficient to reduce the titer of ananti-HTLV-1 antibody in the serum to 50%, 25%, 10% or 5% of the titerpresent prior to the start of treatment. In some instances an effectiveamount of a composition of the invention is an amount that is sufficientto reduce the titer of an anti-HTLV-1 antibody in the serum to a levelwhich is essentially undetectable. The invention includes a method ofreducing the titer of an anti-HTLV-1 antibody in the serum of a patientinfected with HTLV-1, comprising administering to the patient acomposition of the invention in an amount effective to reduce the titerof the anti-HTLV-1 antibody in the serum compared to that present priorto the start of treatment.

Human Papillomavirus

In another aspect the invention provides a method of treating a patientinfected with a Human Papillomavirus (HPV), or a disease or conditionassociated with HPV infection, such as, for example, warts of the handsand feet, or lesions of the mucous membranes of the oral, anal andgenital cavities. While some types of HPV are relatively harmless, othertypes are spread through sexual contact and give rise to genital orvenereal warts (termed condylomata acuminata) which may give rise tocervical cancer and other genital cancers. The method comprisesadministering to the patient infected with HPV an effective amount of acomposition of the invention comprising one or more polypeptide orconjugate of the invention. The invention also provides a compositionfor use in treating a patient infected with HPV, or a disease orcondition associated with HPV infection, the composition comprising oneor more polypeptide or conjugate of the invention and a pharmaceuticallyacceptable carrier or excipient. A patient diagnosed with HPV infectionincludes a patient exhibiting HPV viral DNA in biopsied tissue (such asgenital tissue), and sometimes (but not always) exhibiting visiblelesions, e.g. on genital tissues.

A composition comprising a polypeptide of the invention will generallybe administered at a dose and frequency similar to what is employed inHPV therapeutic regimens using clinically-approved interferon-alphapolypeptides, such as, for example, INTRON® A (Interferon alfa-2b,recombinant; Schering Corporation). A recommended dose of INTRON A forthe treatment of condylomata acuminata is 1.0 million IU injected intoeach lesion, for up to 5 lesions, using a tuberculin or similar syringeand a 25- to 30-gauge needle, three times per week on alternate days,for 3 weeks. Patients with 6 to 10 Condylomata may receive a second(sequential) course of treatment at the above dosage schedule, to treatup to five additional condylomata per course of treatment. Patients withgreater than 10 Condylomata may receive additional sequences dependingon how large a number of condylomata are present. The interferon mayalternatively or in addition be applied topically, e.g. in a cream orointment form (as described for example in Stentella et al. (1996) Clin.Exp. Obstet. Gynecol. 23(1):29-36). Such dosing schedules provide usefulranges for dosage of a polypeptide of the invention for the treatment ofHPV infection, or a disease or condition associated with HPV infectionsuch as condylomata acuminata. Depending on a number of factors(including but not limited to the activity and the pharmacokinetics ofthe polypeptide of the invention and the size, age and health of thepatient), the polypeptide of the invention may be administered in loweramounts and/or less frequently than described above. Likewise, acomposition comprising a conjugate of the invention will generally beadministered, e.g. intralesionally or topically, at a dose effective toreduce the amount of HPV viral DNA in the effected tissues or to reducethe size/or number of genital lesions in the infected individual.

In some instances the polypeptide or conjugate of the invention isadministered in combination with one or more additional therapeuticagent(s). For example, the polypeptide or conjugate of the invention maybe administered in combination with an anti-HPV therapeutic such asPodofilox (Condylox) and/or Podophyllin (Pododerm, Podocon-25).

The precise amount and frequency of administration of the polypeptide orconjugate of the invention will depend on a number of factors such asthe specific activity and the pharmacokinetic properties of thepolypeptide or the conjugate, as well as the nature of the conditionbeing treated, among other factors known to those of skill in the art.Normally, the dose should be capable of preventing or lessening theseverity or spread of the indication being treated. Such a dose may betermed an “effective” or “therapeutically effective” amount. It will beapparent to those of skill in the art that an effective amount of apolypeptide, conjugate or composition of the invention depends, interalia, upon the condition being treated, the dose, the administrationschedule, whether the polypeptide or conjugate or composition isadministered alone or in combination with other therapeutic agents, theserum half-life and other pharmacokinetic properties of the polypeptide,conjugate or composition, as well as the size, age, and general healthof the patient. The dosage and frequency of administration isascertainable by one skilled in the art using known techniques.

The effectiveness of treatment may be determined by measuring the HPVviral load, such as, for example, measuring the level of HPV viral DNAin biopsied tissue. In some instances, an effective amount of acomposition of the invention is one that is sufficient to achieve areduction in viral load by at least 0.5 log unit, such as at least 1 logunit, at least 2 log units, at least 3 log units, at least 4 log units,at least 5 log units, at least 6 log units, or at least 7 log units overthe course of treatment, compared to the viral load prior to treatment.In some instances an effective amount of a composition of the inventionis an amount that is sufficient to reduce viral load to levels which areessentially undetectable. The invention includes a method of reducingthe level of HPV viral DNA in tissue of a patient infected with HPV,comprising administering to the patient a composition of the inventionin an amount effective to reduce the level of HPV viral DNA compared tothat present prior to the start of treatment.

The effectiveness of treatment may alternatively or in addition bedetermined by observing the size or number of genital lesions(condylomata) in the infected individual. In some instances an effectiveamount of a composition of the invention is an amount that is sufficientto reduce the size and/or number of condylomata in the infectedindividual. The invention includes a method of reducing reduce the sizeand/or number of condylomata in a patient infected with HPV, comprisingadministering to the patient a composition of the invention in an amounteffective to reduce the size and/or number of condylomata in the patientcompared to those present prior to the start of treatment.

Formulations and Routes of Administration

Therapeutic formulations of the polypeptide or conjugate of theinvention are typically administered in a composition that includes oneor more pharmaceutically acceptable carriers or excipients. Suchpharmaceutical compositions may be prepared in a manner known per se inthe art to result in a polypeptide pharmaceutical that is sufficientlystorage-stable and is suitable for administration to humans or animals.

Drug Form

The polypeptide or conjugate of the invention can be used “as is” and/orin a salt form thereof. Suitable salts include, but are not limited to,salts with alkali metals or alkaline earth metals, such as sodium,potassium, calcium and magnesium, as well as e.g. zinc salts. Thesesalts or complexes may by present as a crystalline and/or amorphousstructure.

Excipients

“Pharmaceutically acceptable” means a carrier or excipient that at thedosages and concentrations employed does not cause any untoward effectsin the patients to whom it is administered. Such pharmaceuticallyacceptable carriers and excipients are well known in the art (seeRemington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed.,Mack Publishing Company (1990); Pharmaceutical Formulation Developmentof Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor &Francis (2000); and Handbook of Pharmaceutical Excipients, 3rd edition,A. Kibbe, Ed., Pharmaceutical Press (2000)).

Mix of Drugs

The composition of the invention may be administered alone or inconjunction with other therapeutic agents. Ribavirin, for example, isoften co-administered with IFN-alpha and has been shown to increaseefficacy in antiviral treatments, such as HCV treatment. A variety ofsmall molecules are being developed against both viral targets (viralproteases, viral polymerase, assembly of viral replication complexes)and host targets (host proteases required for viral processing, hostkinases required for phosphorylation of viral targets such as NS5A andinhibitors of host factors required to efficiently utilize the viralIRES). Other cytokines may be co-administered, such as for example IL-2,IL-12, IL-23, IL-27, or IFN-gamma. These agents may be incorporated aspart of the same pharmaceutical composition or may be administeredseparately from the polypeptide or conjugate of the invention, eitherconcurrently or in accordance with another treatment schedule. Inaddition, the polypeptide, conjugate or composition of the invention maybe used as an adjuvant to other therapies.

Patients

A “patient” for the purposes of the present invention includes bothhumans and other mammals. Thus the methods are applicable to both humantherapy and veterinary applications.

Types of Composition and Administration Route

The pharmaceutical composition comprising the polypeptide or conjugateof the invention may be formulated in a variety of forms, e.g. as aliquid, gel, lyophilized, or as a compressed solid. The preferred formwill depend upon the particular indication being treated and will beapparent to one skilled in the art.

The administration of the formulations of the present invention can beperformed in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, intracerebrally, intranasally,transdermally, intraperitoneally, intramuscularly, intrapulmonary,vaginally, rectally, intraocularly, or in any other acceptable manner.The formulations can be administered continuously by infusion, althoughbolus injection is acceptable, using techniques well known in the art,such as pumps (e.g., subcutaneous osmotic pumps) or implantation. Insome instances the formulations may be directly applied as a solution,cream, ointment, or spray.

Parenterals

An example of a pharmaceutical composition is a solution designed forparenteral administration. Although in many cases pharmaceuticalsolution formulations are provided in liquid form, appropriate forimmediate use, such parenteral formulations may also be provided infrozen or in lyophilized form. In the former case, the composition mustbe thawed prior to use. The latter form is often used to enhance thestability of the active compound contained in the composition under awider variety of storage conditions, as it is recognized by thoseskilled in the art that lyophilized preparations are generally morestable than their liquid counterparts. Such lyophilized preparations arereconstituted prior to use by the addition of one or more suitablepharmaceutically acceptable diluents such as sterile water for injectionor sterile physiological saline solution.

Parenterals may be prepared for storage as lyophilized formulations oraqueous solutions by mixing, as appropriate, the polypeptide having thedesired degree of purity with one or more pharmaceutically acceptablecarriers, excipients or stabilizers typically employed in the art (allof which are termed “excipients”), for example buffering agents,stabilizing agents, preservatives, isotonifiers, non-ionic detergents,antioxidants and/or other miscellaneous additives.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. They are typically present at a concentrationranging from about 2 mM to about 50 mM. Suitable buffering agents foruse with the present invention include both organic and inorganic acidsand salts thereof such as citrate buffers (e.g., monosodiumcitrate-disodium citrate mixture, citric acid-trisodium citrate mixture,citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g.,succinic acid-monosodium succinate mixture, succinic acid-sodiumhydroxide mixture, succinic acid-disodium succinate mixture, etc.),tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaricacid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture,etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture,fumaric acid-disodium fumarate mixture, monosodium fumarate-disodiumfumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodiumglyconate mixture, gluconic acid-sodium hydroxide mixture, gluconicacid-potassium glyuconate mixture, etc.), oxalate buffer (e.g., oxalicacid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture,oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g.,lactic acid-sodium lactate mixture, lactic acid-sodium hydroxidemixture, lactic acid-potassium lactate mixture, etc.) and acetatebuffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodiumhydroxide mixture, etc.). Additional possibilities are phosphatebuffers, histidine buffers and trimethylamine salts such as Tris.

Preservatives are added to retard microbial growth, and are typicallyadded in amounts of about 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, meta-cresol,methyl paraben, propyl paraben, octadecyldimethylbenzyl ammoniumchloride, benzalkonium halides (e.g. benzalkonium chloride, bromide oriodide), hexamethonium chloride, alkyl parabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol and 3-pentanol.

Isotonicifiers are added to ensure isotonicity of liquid compositionsand include polyhydric sugar alcohols, preferably trihydric or highersugar alcohols, such as glycerin, erythritol, arabitol, xylitol,sorbitol and mannitol. Polyhydric alcohols can be present in an amountbetween 0.1% and 25% by weight, typically 1% to 5%, taking into accountthe relative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols(enumerated above); amino acids such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine,2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugaralcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol,xylitol, ribitol, myoinisitol, galactitol, glycerol and the like,including cyclitols such as inositol; polyethylene glycol; amino acidpolymers; sulfur-containing reducing agents, such as urea, glutathione,thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglyceroland sodium thiosulfate; low molecular weight polypeptides (i.e. <10residues); proteins such as human serum albumin, bovine serum albumin,gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructoseand glucose; disaccharides such as lactose, maltose and sucrose;trisaccharides such as raffinose, and polysaccharides such as dextran.Stabilizers are typically present in the range of from 0.1 to 10,000parts by weight based on the active protein weight.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe present to help solubilize the therapeutic agent as well as toprotect the therapeutic polypeptide against agitation-inducedaggregation, which also permits the formulation to be exposed to shearsurface stress without causing denaturation of the polypeptide. Suitablenon-ionic surfactants include polysorbates (20, 80, etc.), polyoxamers(184, 188 etc.), Pluronic® polyols, polyoxyethylene sorbitan monoethers(Tween®-20, Tween®-80, etc.).

Additional miscellaneous excipients include bulking agents or fillers(e.g. starch), chelating agents (e.g. EDTA), antioxidants (e.g.,ascorbic acid, methionine, vitamin E) and cosolvents.

The active ingredient may also be entrapped in microcapsules prepared,for example, by coascervation techniques or by interfacialpolymerization, for example hydroxymethylcellulose, gelatin orpoly-(methylmethacylate) microcapsules, in colloidal drug deliverysystems (for example liposomes, albumin microspheres, microemulsions,nano-particles and nanocapsules) or in macroemulsions. Such techniquesare disclosed in Remington's Pharmaceutical Sciences, supra.

In one aspect of the invention the composition is a liquid composition,such as an aqueous composition, and comprises a sulfoalkyl ethercyclodextrin derivative of the formula

whereinn is 4, 5 or 6; R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, and R₉ are each,independently, —O— or a —O—(C₂-C₆ alkyl)-SO₃— group, wherein at leastone of R₁, R₂ or R₃ is independently a —O—(C₂-C₆ alkyl)-SO₃— group; andS₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ are each, independently, apharmaceutically acceptable cation, including H⁺.

It should be noted that when n=4, the sulfoalkyl ether cyclodextrin mayalso be referred to as a α-sulfoalkyl ether cyclodextrin. In a similarway, when n=5, the term β-sulfoalkyl ether cyclodextrin may be employedand when n=6, the sulfoalkyl ether cyclodextrin may also be referred toas a γ-sulfoalkyl ether cyclodextrin.

In a further embodiment, n is 5 or 6. In a preferred embodiment n=6.

In a still further embodiment R₁, R₂ or R₃ is independently selectedfrom the group consisting of —OCH₂CH₂CH₂SO₃—, —OCH₂CH₂CH₂CH₂SO₃— and—OCH₂CH₂CH₂CH₂CH₂SO₃—. Most preferably, R₁, R₂ or R₃ is independently—OCH₂CH₂CH₂CH₂SO₃—.

In a further embodiment S₁, S₂, S₃, S₄, S₅, S₆, S₇, S₈, and S₉ are each,independently, a pharmaceutically acceptable cation selected from H⁺,alkali metals (e.g. Li⁺, Na⁺, K⁺), alkaline earth metals (e.g., Ca⁺²,Mg⁺²), ammonium ions and amine cations such as the cations of (C₁-C₆)alkylamines, piperidine, pyrazine, (C₁-C₆) alkanolamine and(C₄-C₈)cycloalkanolamine. Most preferably, S₁, S₂, S₃, S₄, S₅, S₆, S₇,S₈, and S₉ are each, independently, a pharmaceutically acceptable cationselected from the group consisting of H⁺, Li⁺, Na⁺, K⁺, in particularNa⁺.

The sulfoalkyl ether cyclodextrin may contain from 1 to 18 sulfoalkylgroups (when n=4), from 1-21 sulfoalkyl groups (when n=5) or from 1-21(when n=6). In a preferred embodiment of the invention n=5 and thesulfoalkylether derivative comprises, on average, 2-20 sulfoalkyl groups(in particular sulfobutyl groups), such as 3-10 sulfoalkyl groups (inparticular sulfobutyl groups), more preferably 4-9 sulfoalkyl groups (inparticular sulfobutyl groups), even more preferably 5-9 sulfoalkylgroups (in particular sulfobutyl groups), such as 6-8 sulfoalkyl groups(in particular sulfobutyl groups), e.g. 7 sulfoalkyl groups (inparticular sulfobutyl groups).

In some instances the sulfoalkyl ether cyclodextrin derivative is asalt, in particular a sodium salt, of β-cyclodextrin sulfobutyl ether(i.e. n=5), which on average contains 7 sulfobutyl groups. Thissulfoalkyl ether cyclodextrin derivative is also termed SBE7-β-CD and isavailable as Captisol® (Cydex, Overland Park, Kans.).

The term “C₁-C₆ alkyl” represents a branched or straight alkyl grouphaving from one to six carbon atoms. Typical C₁-C₆ alkyl groups include,but are not limited to, methyl ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, sec-butyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl andiso-hexyl.

The term “C₂-C₆ alkyl” represents a branched or straight alkyl grouphaving from two to six carbon atoms. Typical C₂-C₆ alkyl groups include,but are not limited to, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, tert-butyl, n-pentyl, iso-pentyl, n-hexyl and iso-hexyl.

Further details concerning compositions comprising the polypeptidesdisclosed herein and sulfoalkyl ether cyclodextrin derivatives can befound in WO 03/002152, particularly the section entitled “The sulfoalkylether cyclodextrin derivative” on pp. 37-49, incorporated herein byreference.

Parenteral formulations to be used for in vivo administration must besterile. This is readily accomplished, for example, by filtrationthrough sterile filtration membranes.

Sustained Release Preparations

Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing thepolypeptide or conjugate, the matrices having a suitable form such as afilm or microcapsules. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate) orpoly(vinylalcohol)), polylactides, copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the ProLease® technology orLupron Depot® (injectable microspheres composed of lactic acid-glycolicacid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyricacid. While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for long periods such asup to or over 100 days, certain hydrogels release proteins for shortertime periods. When encapsulated polypeptides remain in the body for along time, they may denature or aggregate as a result of exposure tomoisture at 37° C., resulting in a loss of biological activity andpossible changes in immunogenicity. Rational strategies can be devisedfor stabilization depending on the mechanism involved. For example, ifthe aggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Oral Administration

For oral administration, the pharmaceutical composition may be in solidor liquid form, e.g. in the form of a capsule, tablet, suspension,emulsion or solution. The pharmaceutical composition is preferably madein the form of a dosage unit containing a given amount of the activeingredient. A suitable daily dose for a human or other mammal may varywidely depending on the condition of the patient and other factors, butcan be determined by persons skilled in the art using routine methods.

Solid dosage forms for oral administration may include capsules,tablets, suppositories, powders and granules. In such solid dosageforms, the active compound may be admixed with at least one inertdiluent such as sucrose, lactose, or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances, e.g. lubricatingagents such as magnesium stearate. In the case of capsules, tablets andpills, the dosage forms may also comprise buffering agents. Tablets andpills can additionally be prepared with enteric coatings.

The polypeptides or conjugates may be admixed with adjuvants such aslactose, sucrose, starch powder, cellulose esters of alkanoic acids,stearic acid, talc, magnesium stearate, magnesium oxide, sodium andcalcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodiumalginate, polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tabletedor encapsulated for conventional administration. Alternatively, they maybe dissolved in saline, water, polyethylene glycol, propylene glycol,ethanol, oils (such as corn oil, peanut oil, cottonseed oil or sesameoil), tragacanth gum, and/or various buffers. Other adjuvants and modesof administration are well known in the pharmaceutical art. The carrieror diluent may include time delay material, such as glycerylmonostearate or glyceryl distearate alone or with a wax, or othermaterials well known in the art.

The pharmaceutical compositions may be subjected to conventionalpharmaceutical operations such as sterilization and/or may containconventional adjuvants such as preservatives, stabilizers, wettingagents, emulsifiers, buffers, fillers, etc., e.g. as disclosed elsewhereherein.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants such as wetting agents,sweeteners, flavoring agents and perfuming agents.

Pulmonary Delivery

Formulations suitable for use with a nebulizer, either jet orultrasonic, will typically comprise the polypeptide or conjugatedissolved in water at a concentration of, e.g., about 0.01 to 25 mg ofconjugate per mL of solution, preferably about 0.1 to 10 mg/mL. Theformulation may also include a buffer and a simple sugar (e.g., forprotein stabilization and regulation of osmotic pressure), and/or humanserum albumin ranging in concentration from 0.1 to 10 mg/ml. Examples ofbuffers that may be used are sodium acetate, citrate and glycine.Preferably, the buffer will have a composition and molarity suitable toadjust the solution to a pH in the range of 3 to 9. Generally, buffermolarities of from 1 mM to 50 mM are suitable for this purpose. Examplesof sugars which can be utilized are lactose, maltose, mannitol,sorbitol, trehalose, and xylose, usually in amounts ranging from 1% to10% by weight of the formulation.

The nebulizer formulation may also contain a surfactant to reduce orprevent surface induced aggregation of the protein caused by atomizationof the solution in forming the aerosol. Various conventional surfactantscan be employed, such as polyoxyethylene fatty acid esters and alcohols,and polyoxyethylene sorbitan fatty acid esters. Amounts will generallyrange between 0.001% and 4% by weight of the formulation. An especiallypreferred surfactant for purposes of this invention is polyoxyethylenesorbitan monooleate.

Specific formulations and methods of generating suitable dispersions ofliquid particles of the invention are described in WO 94/20069, U.S.Pat. No. 5,915,378, U.S. Pat. No. 5,960,792, U.S. Pat. No. 5,957,124,U.S. Pat. No. 5,934,272, U.S. Pat. No. 5,915,378, U.S. Pat. No.5,855,564, U.S. Pat. No. 5,826,570 and U.S. Pat. No. 5,522,385 which arehereby incorporated by reference.

Formulations for use with a metered dose inhaler device will generallycomprise a finely divided powder. This powder may be produced bylyophilizing and then milling a liquid conjugate formulation and mayalso contain a stabilizer such as human serum albumin (HSA). Typically,more than 0.5% (w/w) HSA is added. Additionally, one or more sugars orsugar alcohols may be added to the preparation if necessary. Examplesinclude lactose maltose, mannitol, sorbitol, sorbitose, trehalose,xylitol, and xylose. The amount added to the formulation can range fromabout 0.01 to 200% (w/w), preferably from approximately 1 to 50%, of theconjugate present. Such formulations are then lyophilized and milled tothe desired particle size.

The properly sized particles are then suspended in a propellant with theaid of a surfactant. The propellant may be any conventional materialemployed for this purpose, such as a chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,including trichlorofluoromethane, dichlorodifluoromethane,dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, orcombinations thereof. Suitable surfactants include sorbitan trioleateand soya lecithin. Oleic acid may also be useful as a surfactant. Thismixture is then loaded into the delivery device. An example of acommercially available metered dose inhaler suitable for use in thepresent invention is the Ventolin metered dose inhaler, manufactured byGlaxo Inc., Research Triangle Park, N.C., USA.

Formulations for powder inhalers will comprise a finely divided drypowder containing conjugate and may also include a bulking agent, suchas lactose, sorbitol, sucrose, or mannitol in amounts which facilitatedispersal of the powder from the device, e.g., 50% to 90% by weight ofthe formulation. The particles of the powder shall have aerodynamicproperties in the lung corresponding to particles with a density ofabout 1 g/cm² having a median diameter less than 10 micrometers,preferably between 0.5 and 5 micrometers, most preferably of between 1.5and 3.5 micrometers. An example of a powder inhaler suitable for use inaccordance with the teachings herein is the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass., USA.

The powders for these devices may be generated and/or delivered bymethods disclosed in U.S. Pat. No. 5,997,848, U.S. Pat. No. 5,993,783,U.S. Pat. No. 5,985,248, U.S. Pat. No. 5,976,574, U.S. Pat. No.5,922,354, U.S. Pat. No. 5,785,049 and U.S. Pat. No. 5,654,007.

Mechanical devices designed for pulmonary delivery of therapeuticproducts, include but are not limited to nebulizers, metered doseinhalers, and powder inhalers, all of which are familiar to those ofskill in the art. Specific examples of commercially available devicessuitable for the practice of this invention are the Ultravent nebulizer,manufactured by Mallinckrodt, Inc., St. Louis, Mo., USA; the Acorn IInebulizer, manufactured by Marquest Medical Products, Englewood, Colo.,USA; the Ventolin metered dose inhaler, manufactured by Glaxo Inc.,Research Triangle Park, N.C., USA; the Spinhaler powder inhaler,manufactured by Fisons Corp., Bedford, Mass., USA the “standing cloud”device of Nektar Therapeutics, Inc., San Carlos, Calif., USA; the AIRinhaler manufactured by Alkermes, Cambridge, Mass., USA; and the AERxpulmonary drug delivery system manufactured by Aradigm Corporation,Hayward, Calif., USA.

Kits

The present invention also provides kits including the polypeptides,conjugates, polynucleotides, expression vectors, cells, methods,compositions, and systems, and apparatuses of the invention. Kits of theinvention optionally comprise at least one of the following of theinvention: (1) an apparatus, system, system component, or apparatuscomponent as described herein; (2) at least one kit component comprisinga polypeptide or conjugate or polynucleotide of the invention; a plasmidexpression vector encoding a polypeptide of the invention; a cellexpressing a polypeptide of the invention; or a composition comprisingat least one of any such component; (3) instructions for practicing anymethod described herein, including a therapeutic or prophylactic method,instructions for using any component identified in (2) or anycomposition of any such component; and/or instructions for operating anyapparatus, system or component described herein; (4) a container forholding said at least one such component or composition, and (5)packaging materials.

In a further aspect, the present invention provides for the use of anyapparatus, component, composition, or kit described above and herein,for the practice of any method or assay described herein, and/or for theuse of any apparatus, component, composition, or kit to practice anyassay or method described herein.

EXAMPLES

The following examples are offered to illustrate the present invention,but not to limit the spirit or scope of the present invention in anyway.

Materials and Methods

I. Determination of Surface-Accessible Residues

Accessible Surface Area (ASA)

The computer program Access (B. Lee and F. M. Richards, J. Mol. Biol.55: 379-400 (1971)) version 2 (©1983 Yale University) was used tocompute the accessible surface area (ASA) of the individual atoms in thestructure. This method typically uses a probe-size of 1.4 Å and definesthe Accessible Surface Area (ASA) as the area formed by the center ofthe probe. Prior to this calculation all water molecules and allhydrogen atoms should be removed from the coordinate set, as shouldother atoms not directly related to the protein.

Fractional ASA of Side Chain

The fractional ASA of the side chain atoms is computed by division ofthe sum of the ASA of the atoms in the side chain by a valuerepresenting the ASA of the side chain atoms of that residue type in anextended ALA-x-ALA tripeptide. See Hubbard, Campbell & Thornton (1991)J. Mol. Biol. 220, 507-530. For this example the CA atom is regarded asa part of the side chain of glycine residues but not for the remainingresidues. The following values are used as standard 100% ASA for theside chain (Table 6):

Ala  69.23 Å² Leu 140.76 Å² Arg 200.35 Å² Lys 162.50 Å² Asn 106.25 Å²Met 156.08 Å² Asp 102.06 Å² Phe 163.90 Å² Cys  96.69 Å² Pro 119.65 Å²Gln 140.58 Å² Ser  78.16 Å² Glu 134.61 Å² Thr 101.67 Å² Gly  32.28 Å²Trp 210.89 Å² His 147.00 Å² Tyr 176.61 Å² Ile 137.91 Å² Val 114.14 Å²

Residues not detected in the structure are defined as having 100%exposure as they are thought to reside in flexible regions. In the casewhere an ensemble of NMR structures is analyzed, the average ASA valueof the ensemble is used.

Determination of Surface Exposed Residues when No Three-DimensionalStructure is Available:

When no three-dimensional structure is available or if the structure isnot detailed enough to determine surface accessibility (e.g. if only theposition of the CA atoms is known) the surface accessibility may beinferred from a sequence alignment created as follows:

-   -   A: If the structure is known but not detailed enough to        determine surface accessibility:        -   The low detail structure is included in a structure-based            sequence alignment to the known structures of the sequence            family using the MODELER program available from Molecular            Simulations, Inc.    -   B: If no structure is known:        -   The sequence is aligned to a predefined sequence alignment,            including the sequences of the known structures of the            sequence family, that may be prepared using the            “profile/structure alignment” option of the program ClustalW            (Thompson et al. (1994) Nucleic Acids Research            22:4673-4680).

From the sequence alignment obtained in A or B, residues in the sequenceto be analyzed at positions equivalent to residues exposed in at leastone of the other sequences having a known structure are defined as beingexposed. The degree of exposure is taken to be the largest value for theequivalent residues in the other sequences. In cases where the sequenceto be analyzed is at an insertion (i.e. there are no equivalent residuesin the other sequences) this residue is defined as being fully exposed,as it most probably is located in a turn/loop region. In cases where alow detailed structure exists, those residues not observed in thestructure are defined as being fully exposed, as they are thought to bein flexible regions.

Determining Distances Between Atoms:

The distance between atoms is readily determined using moleculargraphics software, e.g. InsightII® 98.0 from Molecular Simulations, Inc.

II. Protein Expression and Purification

A. Expression and Purification from CHO Cells

Some polypeptides of the invention were produced in Chinese HamsterOvary (CHO) K1 cells (ATCC: CCL-61) that were stably transfected andselected with G418 to establish clonal cell lines.

1. CHO Expression Construct

Nucleic acids encoding polypeptides of the invention were cloned into aCHO expression vector, under control of the SV40 promoter and in-framewith a sequence which encodes an N-terminal leader sequence, and,optionally, one or two a C-terminal tag sequences. The leader sequencewas either a generic leader sequence, IFN alpha 6 leader or a modifiedIFN alpha 6 leader sequence, and C-terminal tags included an E-tag(Amersham Biosciences) &/or a His-tag. Plasmid production was inXL1-Blue cells.

2. Selection of Stable Subclones Expressing IFN-Alpha Polypeptides:

Materials:

Culture medium: DMEM-F12 with G418, FBS and Penicillin, Streptomycin andGlutamine (PSG; Gibco/Invitrogen);

1×PBS (Gibco/Invitrogen); Trypsin/EDTA

Anti E-Tag Antibody-HRP conjugate (Amersham BioSciences)

ECL Plus Western Blotting detection Reagents (Amersham BioSciences)

Procedure: Stable transfectants were generated under selection with G418in DMEM/F-12 medium with FBS and penicillin. Cells were split into T175flasks with 50 ml of selection medium and incubated in a 37° C. CO₂incubator for ˜24 hr. or until cells reached 80% confluence. Cells wereharvested by washing with PBS followed by addition of 2.5 mlTrypsin/EDTA and incubation at 37° C. for 3-5 min. Cells were collectedand recovered by centrifugation at 1000 g for 30 min in a Beckman Modelbench top centrifuge. Cells were washed once in PBS and resuspended in 3ml PBS with 1% FBS. The cell density was determined and adjusted to1×10⁶ cell/ml with PBS/FBS. For each IFN-alpha, polypeptide cells weresorted in a DakoCytomation MoFlo sorter into 2-5 96 well platescontaining 200 ml of selection medium. The plates were incubated in a37° C. incubator for 10-14 days to allow the sorted cells to grow. Twosubclones were selected for each IFN-alpha polypeptide for high levelexpression first by dot blot analysis and subsequently confirmed byWestern blot analysis using an anti-E tag antibody-HRP conjugate andchemiluminescent detection.

3. Protein Expression:

Materials:

DMEM-F12 medium (Gibco/Invitrogen)

Ultra CHO medium (BioWhittaker)

CHO III A medium (Gibco/Invitrogen)

Ex-Cyte Growth Enhancing Media supplement (Serologicals Proteins)

ITSA (Insulin, Transferrin, Selenium supplement for adherent culture;Gibco/Invitrogen)

Penicillin/Streptomycin (P/S)

FBS, PBS, Trypsin

Procedure:

Day 1: Cells from one T-175 flask were transferred to one roller bottle(1700 Cm²) in 300 ml DMEM-F12 with 10% FBS and 1× P/S and grown in a 37°C. CO₂ incubator.

Day 3: Medium was changed to 300 ml fresh DMEM-F12-FBS-P/S.

Day 5: The medium was changed to 300 ml Ultra CHO with 1/1000 Ex-Cyteand P/S.

Day 7: The media was replaced with 300 ml CHO III A+P/S productionmedium.

Supernatants were harvested on Day 8, 9 and 10. The supernatants werecentrifuged at 2000 g for 20 min in a Beckman Coulter Allegra 6R benchtop centrifuge and filtered using a 0.2μ PES bottle top sterile filterand stored at 4° C. for purification.

4. Protein Purification:

Some polypeptides of the invention were expressed as fusion proteinscontaining a 13 amino acid E-tag sequence at the C-terminus. Suchpolypeptides were purified using an E-tag affinity column, as follows.

Materials: Recombinant Phage Antibody System Purification Module(Amersham BioSciences, Cat. No. 17-1362-01). Purification kit contains a16 mm diameter×25 mm height (5 ml bed volume) anti E-Tag column andassociated buffers.

Procedure: Supernatants collected from CHO-HK1 cells in roller bottleswere clarified using a combination of centrifugation at 2800×g for 20min and filtration using a 0.2t PES bottle top filter module.Supernatants were loaded onto the E-tag column equilibrated in RPASbinding buffer at 150 Cm/h (5 ml/min). The column was washed with 5 CV(column volume) of binding buffer and the protein was eluted at 75 Cm/h(2.5 ml/min) with RPAS elution buffer. Elution fractions wereneutralized with 0.05 volumes of 1M Tris-Cl pH 8.0, dialyzed into PBS,concentrated to 0.1-1.5 mg/ml and stored frozen in aliquots at −80° C.Samples for assays were formulated at 50 μg/ml in PBS with 0.5% BSA andstored frozen in aliquots at −80° C. Samples were routinely analyzed bySDS-PAGE followed by Coomassie staining using materials, reagents andprotocols obtained from Invitrogen. Protein concentrations wereroutinely determined by the BCA assay using an IFN-alpha standard, theconcentration of which had been verified by amino acid analysis.

B. Expression and Purification from E. coli

Some polypeptides of the invention were produced in E. coli as inclusionbodies, which were purified and refolded as follows.

1. E. coli Expression Construct

In some instances, nucleic acids encoding polypeptides of the inventionwere modified for improved expression in E. coli. Such modificationscomprised replacing rare Arg-Arg codon pairs AGGAGG at nucleotidepositions 34-39, and AGGAGA at nucleotide positions 64-69 (positionnumbering relative to SEQ ID NO:18), each with Arg-Arg codon pairs suchas CGTCGC which are preferred in E. coli, and adding a methionine codon(ATG) to the 5′ end of the coding sequence. The coding sequence wasplaced into the pET-42 expression vector (Novagen) under control of a T7promoter with kanamycin selection marker, or into the pQE80-Kanexpression vector (Qiagen) under the control of a T5 promoter withkanamycin selection marker.

2. Protein Expression

pET-42 vectors containing interferon coding sequences were transformedinto an E. coli strain such as BL21(DE3) using standard methods, andplated on to agar plates containing 50 μg/ml kanamycin and incubated at37° C. After 18-24 h, three separate colonies were picked andtransferred into tubes containing 5 ml of 2×YT with 50 μg/ml kanamycinand incubated overnight at 37° C. The overnight culture was used toinoculate 2 sets of flasks containing 100 ml of 2×YT with 50 μg/mlkanamycin. The growth of the culture at 37° C. was monitored at OD₆₀₀.The culture was induced at an OD₆₀₀ of 0.5-0.8 with 1 mM IPTG for 3 h at37° C. IPTG induced cultures were analyzed for expression by SDS-PAGE bylysing pelleted cells in SDS sample buffer. The corresponding uninducedsets of cultures were used to prepare frozen stocks by addition of 25%glycerol and freezing cells in 1 ml aliquots at −80° C. pQE80-Kanvectors containing interferon coding sequences were transformed into E.coli strains W3110 or W3110-fhuA. Expression was verified as describedabove.

Larger scale shake flask expression was performed by inoculating 4×1 L2×YT media+kanamycin with 25 ml of an overnight culture. Cultures weremonitored at OD600 and induced with 1 mM IPTG at 0.5-0.8 OD units. After3 h of induction cells are harvested by centrifugation at 5000 g andstored frozen at −80° C. Cells were disrupted using 2-3 passes through aFrench press or a APV 1000 homogenizer at 10,000 psi and processed asdescribed under “Isolation of IB” and subsequent sections.

Fed-batch fermentation was conducted at 10 L scale in a B. Braunbioreactor in Terrific Broth (TB) medium supplemented with trace elementsolution and 40 mg/L kanamycin. Fermentation was initiated byinoculating the bioreactor with a 400 ml overnight culture in TB medium.During the initial growth phase the dissolved oxygen (DO) was maintainedat 50% by varying the agitation rate. When the OD600 of the culturereached 5.0, the glycerol/amino acid feed was initiated at 0.5 ml/minand the agitation was set to 1000 rpm. The feed rate was adjusted tomaintain the DO at 40% for the rest of the fermentation process. Whenthe OD600 reached 25 the culture was induced by addition of IPTG to afinal concentration of 1 mM. Three hours post induction the cells wereharvested by centrifugation at 10000×g in a Beckman centrifuge and thecell paste was stored at −60° C. The OD600 at harvest was typicallyaround 35-40.

3. Isolation, Solubilization, Sulfonation and Refolding of InclusionBodies (IB)

For isolating IB the thawed cell paste was resuspended in 1×PBS at 10 mlper gram of cell paste and mixed until a uniform slurry was obtained.The cells were disrupted by two passes through a microfluidizer at17,000-19,000 psi. The cell lysate was adjusted to 1% Triton-X100, mixedfor 10 min and the IB were recovered by centrifugation at 10,000 g for60 min at 2-8° C. The IB pellet was washed once by resuspending in PBSwith 1% Triton-X100 and recovered by centrifugation as above and storedfrozen at −80° C.

For solubilizing IB, the IB pellet was resuspended in Urea buffer (50 mMTris-Cl, 200 mM NaCl, 8 M urea, 2 mM DTT, 1 mM EDTA, pH 8.0) at 10 mlbuffer per gram of cell paste. The suspension was mixed for 30 min andcentrifuged at 10,000 g for 30 min at 2-8° C. The pellet was washed onceby resuspending in the Urea buffer without DTT, centrifuged as above andwashed twice with water. The washed pellet was solubilized in Guanidinebuffer (50 mM Tris-Cl, 200 mM NaCl, 8 M guanidine-HCl, 1 mM EDTA, pH8.0) at 10 ml buffer per gram pellet, mixed for 30-60 min andcentrifuged at 10,000 g for 30 min at 2-8° C. The supernatant containingthe solubilized IFN was adjusted to 10 mg/ml sodium sulfite and 5 mg/mlsodium tetrathionate to initiate the sulfonation process which wasperformed at 2-8° C. for 16 hours. Post-sulfonation the IFN solution wasdiluted 2-fold with water and the IFN pellet was recovered bycentrifugation at 10,000 g at 2-8° C. The pellet was washed twice withwater and resuspended in Guanidine buffer as above. The proteinconcentration of the sulfonated IFN was determined by absorbance at 280nm.

The refolding process was initiated by diluting the sulfonated IFN inGuanidine buffer at 2-8° C. to a final concentration of 100 mg/ml inRefolding buffer (50 mM Tris-Cl, 20 mM NaCl, 2 mM reduced glutathione, 1mM oxidized glutathione). Refolding was performed at 2-8° C. with slowmixing for 6-8 h, followed by addition of CuSO₄ to a final concentrationof 2 mM followed by an additional refolding period of 16-20 h. Theprogress of the refolding reaction was monitored by SDS-PAGE and reversephase HPLC.

4. Purification

The refolded IFN was purified using three chromatography steps. Therefolding solution was adjusted to 80% ammonium sulfate (weight/volume),filtered through a 0.2 μM filter and loaded at 200 Cm/h onto a 20 mlButyl Sepharose Fast Flow Hydrophobic Interaction Chromatography (HIC)column (Amersham Biosciences) equilibrated in Equil buffer (50 mMTris-Cl, 0.8 M ammonium sulfate, pH 8.0). The HIC column was washed with8 Column volumes (CV) of Equil buffer, followed by 8 CV of Wash buffer(50 mM Tris-Cl, 0.5 M ammonium sulfate, pH 8.0). IFN was eluted with 15%ammonium sulfate in 50 mM Tris-Cl, pH 8.0.

The HIC pool was adjusted to 50 mM sodium acetate using 0.5 M sodiumacetate, pH 4.5 stock and diluted two fold. This adjusted pool wasloaded at 90 Cm/h on to a 5 ml HiTrap CM Sepharose Fast Flow column(Amersham Biosciences), equilibrated in 50 mM sodium acetate, 100 mMNaCl, pH 5.0. Post loading the column was washed with 5 CV underequilibration buffer conditions and IFN was eluted using a 20 CVgradient from 100-650 mM NaCl. Peak fractions containing IFN were pooledbased on absorbance at 280 nm.

The CM Sepharose pool was adjusted to 20 mM 1,3 diaminopropane using a 2M1,3 diaminopropane stock to set pH at ˜10. The sample was loaded at 200Cm/h on to a 5 ml HiTrap Q Sepharose Fast Flow column (AmershamBiosciences), equilibrated in 50 mM 1,3 diaminopropane, 100 mM NaCl, pH10.0. Post loading the column was washed with 5 CV of buffer underequilibration conditions and IFN was eluted using a 20 CV gradient from100-500 mM NaCl. The fractions containing IFN are pooled based onabsorbance at 280 nm and immediately dialyzed against 250-500 volumes of50 mM sodium acetate, 150 mM NaCl, pH 5.0 or 50 mM sodium borate, pH9.0. Post-dialysis the IFN samples were sterile filtered using a 0.2 μMfilter in a biosafety cabinet, the concentration was measured byabsorbance at 280 nm and the samples are stored at 2-8° C. for periodsup to a week or in aliquots at −80° C. for extended storage. Foractivity assays the sample was generally formulated in 0.5% BSA, PBS pH7.4 at 5, 20 and 50 ug/ml, and stored frozen in aliquots at −80° C.

III. Activity Assays

A. EMCV-HuH7 Antiviral Assay

Provided below is an exemplary assay for antiviral activity ofinterferon-alpha polypeptides and conjugates of the invention. The assayis a cell-based dose-response assay used to assess the anti-viralpotency of a drug, and is sometimes referred to as “protection fromcytopathic effect” (or PCPE) assay. Briefly, cells are incubated withdrug and exposed to virus. In the absence of drug, cells exposed tovirus die. With increasing concentrations of drug, an increasingproportion of cells survive. The number of surviving cells can bemeasured directly (e.g., by visual counts) or indirectly by estimatingmetabolic rate. For example, metabolic dyes such as MTT or WST-1 may beused as an indirect measure of cell survival. Live cells metabolize suchdyes to form metabolic products which can be quantified byspectrophotometry (optical density).

Materials:

Cells:

HuH7 cells: Human hepatoma cell line (obtained from Dr. Michael Lai,USC-Surgery Department, Los Angeles, Calif.). The cell line may also beobtained from the Cell Bank of the Japanese Collection of ResearchBioresources (JCRB)/Health Science Research Resources Bank (HSRRB),Osaka, Japan. The HuH7 cell line was originally established in thelaboratory of Dr. J. Sato (Okayama University School of Medicine) from a57-year old Japanese male with well-differentiated hepatocellularcarcinoma (Nakabayashi, H., et al. (1982) Cancer Res. 42(9):3858-63).The cell line is negative for Hepatitis B surface antigen.

VERO Cells: African green monkey kidney cell line (ATCC # CCL-81)

L-929 cells: Murine fibroblast cell line (ATCC # CCL-1)

Virus:

Encephalomyocarditis virus (EMCV): tissue culture adapted strain (ATCC #VR-129B). High titer viral stocks were produced in-house by passage inVERO cells

Complete Media:

Dulbecco's Modified Eagle Medium (DMEM, Gibco Cat. No. 11965-092)

10% Fetal Bovine Serum (FBS, Hyclone Cat. No. SH30071.03)

1× Penicillin-streptomycin (PS, Gibco Cat. No. 15140-122)

Reduced Serum Media:

Dulbecco's Modified Eagle Medium (DMEM, Gibco Cat. No. 11965-092)

2% Fetal Bovine Serum (FBS, Hyclone Cat. No. SH30071.03)

1× Penicillin-streptomycin (PS, Gibco Cat. No. 15140-122)

Trypsin/EDTA (Gibco Cat. No. 25300-054)

WST-1 (Roche; Cat. No. 1 644 807)

HuH7 cells were maintained in Complete Media at 37° C. in a humidified5% CO₂ incubator. The cells were harvested with trypsin and split twiceweekly when confluent to a final density of 1-2×10⁶ cells per 25 ml in aT175 flask. One day prior to the assay, the cells were trypsinized andseeded into new T175 flasks at a density of 4.5×10⁶ cells per 25 ml toensure that the cells were in log phase prior to the assay.

Procedure:

A high titer EMCV virus stock was amplified in VERO cells. The lethalconcentration at which 95% of the cells were killed (LC₉₅) wasdetermined by an EMCV viral killing curve on HuH7 cell monolayers.Briefly, HuH7 cells were plated on day one in 96-well microtiter platesat 6×10⁴ cells per well. Virus was serial diluted 1:3 in DMEM+2% FBSwith 10 dilution points and added to the cells on day two. Twenty-fourhours post-infection, cell survival was determined by a tetrazolium saltmetabolism assay, WST-1 (Roche). The LC₉₅ determined for the HuH7-EMCVassay corresponded to an MOI of 0.034 (PFU/cell). Titer of the virusstock was determined by a standard plaque assay on L929 cells.

On day one of the assay, log phase HuH7 cells were harvested withtrypsin, resuspended in Reduced Serum Media and concentrated bycentrifugation. The cell pellets, corresponding to 5 T175 flasks ofcells, were resuspended in 10 ml of Reduced Serum Media, filteredthrough a 40 micron Nylon cell strainer and counted with ahemocytometer. Cell viability was determined by trypan blue exclusion.The cells were resuspended in Reduced Serum Media to a final density of6×10⁵ cells/ml. One hundred microliters of the diluted cells were addedto each well of a 96-well assay plates (6×10⁴ cells/well) and the plateswere incubated at 37° C. in a humidified 5% CO₂ incubator for 4 hours.

The potencies of “reference” interferon alphas and interferon-alphapolypeptides of the invention (also called “test samples”) weredetermined by dose-response analysis. There was generally one testsample and one reference IFN-alpha per plate, each with three replicatecurves of IFN-alpha treated/EMCV challenged cells and two replicatecurves of IFN-alpha treatment alone. The later was assayed to controlfor potential antiproliferative effects of IFN-alpha on HuH7 cells. Thedose-response curves for the reference IFN-alphas generally consisted of8 three-fold dilutions ranging from 100 ng/ml to 0.05 ng/ml. For theIFN-alpha test samples, the three-fold dilutions generally ranged from 5ng/ml to 0.002 ng/ml. Eight wells each of cells treated with virus butno IFN-alpha and cells alone were also run as controls.

The IFN-alpha dilutions were prepared using Reduced Serum Media. Onehundred microliters of the diluted IFN-alpha preparations weretransferred to the assay plates. The assay plates were incubated at 37°C. in a humidified 5% CO₂ incubator for 16 hours.

On day two, the cells were challenged with EMC virus. Medium wasaspirated from each well of the assay plate. The EMCV stock was diluted1:5400 in DMEM+2% FBS. One hundred microliters of the diluted virus,corresponding to 0.034 viral particles per cell, was added to each well.The cells were incubated with virus at 37° C. in a humidified 5% CO₂incubator for 24 hours.

On day three, the number of viable cells in each well was quantified byWST-1 assay. Medium was aspirated from each well of the assay plate. TheWST-1 reagent was diluted 1:20 in Reduced Serum Media, 100 μl of thediluted WST-1 reagent was added to each well and the cells wereincubated at 37° C. in a humidified 5% CO₂ incubator for 60 minutes. Thenumber of viable cells in each well was quantified by measuring OD at450 nm on a plate reader.

Analysis:

The antiviral potency of the IFN-alpha reference and test samples werecalculated with the equation:Antiviral potency=(Viable cells_(C+I+V)−Viable cells_(C+V))/(Viablecells_(C+I)−Viable cells_(C+V))*100%

where C+V=HuH7 cells+EMCV, C+I=HuH7 cells+IFN-α, and C+I+V=HuH7cells+IFN-α+EMCV

Dose-response curves were analyzed by non-linear regression usingGraphPad Prism 4 (GraphPad Software Inc.) The following equation wasused for the curve fits:

$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{1 + 10^{{({{{Log}\;{EC50}} - X})} \cdot {HillSlope}}}}$

Bottom is the Y value at the bottom plateau; Top is the Y value at thetop plateau, and LogEC50 is the X value when the response is halfwaybetween Bottom and Top. The Levenberg-Marquardt method was used asoptimization algorithm.

B. T_(H)1 Differentiation Assay

Provided below is an exemplary assay for T_(H)1 differentiation activityof interferon-alpha polypeptides and conjugates of the invention.

Assay Procedure:

Human buffy coats (25-30 ml) containing leukocytes and erythrocytesprepared from 500 ml blood were collected from Stanford Blood Bank theday of assay initiation and kept at room temperature. Each buffy coatwas carefully transferred to a T75 flask and diluted to 100 ml with PBS.For each buffy coat, 13 ml of Histopaque/Ficoll (Sigma H8889) waspipetted into four 50 ml centrifuge tubes, and 25 ml of diluted bloodsample was carefully overlaid on top of the Histopaque/Ficoll withoutdisrupting the interface. The tubes were then centrifuged (20° C., 2500rpm) for 20 minutes. Using 3 ml plastic transfer pipettes, the topplasma was removed to the mononuclear cell layer, followed by transferof the PBMCs to two 50 ml conical tubes (cells from 2Histopaque/Ficoll/buffy coat tubes to one tube). The PBMCs were thendiluted to 50 ml/tube with PBS and centrifuged (20° C., 1000 rpm) for 10minutes to remove platelets. After removal of PBS, the PBMCs andremaining RBCs were mixed to prevent aggregation. 5 ml of RBC lysisbuffer (ammonium chloride buffer) was added and two tubes of cells werecombined to one tube. Each tube, now containing the total PBMC and RBCisolate from one donor, was incubated at room temperature for 10 min.Potential clots of blood cells were removed by filtering the cells witha cell strainer (70 um, Falcon Cat. No. 2350). PBS was added to a totalvolume of 50 ml followed by centrifugation (20° C., 1000 rpm) for 10min. The cell number was finally counted using a hemocytometer.

Next, a fraction of each PBMC preparation was prestained and analyzed byFACS to select PBMC preparations with a percentage of naïve Th0 cellsabove 15%. Two ml of PBMCs were stained with 20 μl FITC-conjugatedanti-human CD45RA (Pharmigen, Cat. No. 555488), 20 μl Cy-chromeconjugated anti-human CD4 (Pharmigen, Cat. No. 555348), 10 μlPE-conjugated anti-human CD8 (Pharmigen, Cat. No. 555367), 10 μlPE-conjugated anti-human CD14 (Pharmigen, Cat. No. 555398), and 10 μlPE-conjugated anti-human CD20 (Pharmigen, Cat. No. 555623), andincubated on ice for 45 minutes. The cells were washed with PBS,resuspended in 1 ml PBS, and filtered with a 40 μm cell strainer(Falcon, Cat. No. 2340). The percentage of naïve T_(H)0 cells (positiveto CD4 and CD45RA and negative to CD8, CD14, CD20) were quantified byFACS, and PBMC preparations with more than 15% naïve T_(H)0 cells wereselected for the assay.

The selected PBMC preparations were stained with 800 t FITC-conjugatedanti-human CD45RA, 800 μl Cy-chrome conjugated anti-human CD4, 500 μlPE-conjugated anti-human CD8, 200 μl PE-conjugated anti-human CD14, and200 μl PE-conjugated anti-human CD20, and incubated on ice for 60minutes. The cells were washed with PBS, PI was added, and the cellswere diluted with 20 ml/ml PBS followed by filtering with a 40 μm cellstrainer. The cells were FACS sorted, and 1×10⁴ naïve T_(H)0 cells(positive to CD4 and CD45RA and negative to CD8, CD14, CD20) weretransferred by MOFLO into each well of 96 well round bottom plates,containing 160 μl DMEM plus Penicillin-streptomycin plus 2 mM Glutamineand 10% Fetal Bovine Serum (Hyclone Cat. No. SH30071.03).

Twenty μl Dynabeads CD3/CD28 T cell expander (Dynal, Cat. No. 111.32)were added to each well. The stimulatory effect of the Dynabeads wascalibrated prior to the experiment to avoid lot-to-lot variance. Next,20 μl/well of protein samples were added to the assay plates. Generally,concentration ranges for IL-4 and IL-12 standards (obtained from R&DSystems) were from 0.04 pg/ml to 10 ng/ml, and concentration ranges forIFN-alpha test samples and IFN-alpha reference sample were from 0.76pg/ml to 200 ng/ml.

The cells were incubated at 37° C. in a humidified 5% CO₂ incubator for7 days. Supernatants from each well were harvested to determine thedegree of T_(H)1 expansion through quantification of the IFN-γ content,using a standard ELISA.

Analysis:

Response=IFN-γ concentrations in pg/ml.

The following equation was used for curve fitting:

$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{1 + 10^{{({{{Log}\;{EC50}} - X})} \cdot {HillSlope}}}}$

The variable Bottom is the Y value at the bottom plateau; Top is the Yvalue at the top plateau, and LogEC50 is the X value when the responseis halfway between Bottom and Top. The Levenberg-Marquardt method wasused as optimization algorithm.

C. Daudi Antiproliferation Assay

Provided below is an exemplary assay for antiproliferative activity ofinterferon-alpha polypeptides and conjugates of the invention.

Cell Line Maintenance:

Daudi Burkitt's lymphoma cells grown in suspension were maintained inT175 tissue culture flasks, containing 50 ml culture medium (RPMI+10%Fetal Bovine Serum (FBS, Hyclone Cat. No. SH30071.03)+1×Penicillin-streptomycin (PS, Gibco Cat. No. 15140-122)+2 mM Glutamine),at 37° C. in a humidified 5% CO₂ incubator. The cells were split 1:10when confluent.

Assay Procedure:

Daudi cells were spun down and washed with 1×PBS. The cell number wasadjusted to 10⁵ cells/ml. 80 μl culture medium was added to each well in96 well round bottom assay plates followed by transfer of 100 μl cells(10⁴ cells/well) to each well.

Eleven dilutions of the IFN-alpha reference material and IFN-alpha testsamples, ranging from 200 ng/ml to 0.2 pg/ml (4-fold dilutions), wereprepared in dilution plates using culture medium. Twenty t of thediluted IFN-alpha preparations were then transferred to the assayplates.

The cells were incubated at 37° C. in a humidified 5% CO₂ incubator.After 48 hours, 1 μCi of methyl-3H thymidine (Amersham Pharmacia, Cat.No. TRK758) was added to each well followed by incubation for 24 hoursat 37° C. in a humidified 5% CO₂ incubator. The cells were harvested onthe following day and incorporation of thymidine was determined.

Analysis:

The EC₅₀ of the IFN-alpha reference and samples were calculated usingthe equation:

$Y = {{Bottom} + \frac{\left( {{Top} - {Bottom}} \right)}{1 + 10^{{({{{Log}\;{EC50}} - X})} \cdot {HillSlope}}}}$where Bottom is the Y value at the bottom plateau; Top is the Y value atthe top plateau, and LogEC50 is the X value when the response is halfwaybetween Bottom and Top. The Levenberg-Marquardt method was used asoptimization algorithm.

Example 1 Determination of Surface-Accessible Residues ofInterferon-Alphas

Surface Exposure of Human Interferon-α2a Residues:

Based on the 24 NMR structures of human interferon-alpha 2a reported byKlaus et al., J. Mol. Biol., 274: 661-675 (1997), the fractional ASA ofside chains was calculated. The sequence numbering used below is basedon the mature sequence of the human interferon-alpha 2a protein(identified herein as SEQ ID NO:32+R23K). It is noted that thisstructure contains two disulphide bridges involving Cys1-Cys98 andCys29-Cys138, respectively. By computing the ASA and the fractional ASAand taking the average of the 24 structures, focusing on the ASA of theside chains, it was determined that the following residues have morethan 25% fractional ASA: D2, L3, P4, Q5, T6, H7, S8, L9, G10, R12, R13,M16, A19, Q20, R22, K23, I24, S25, L26, F27, S28, L30, K31, R33, H34,D35, G37, Q40, E41, E42, G44, N45, Q46, Q48, K49, A50, E51, E58, Q61,Q62, N65, S68, T69, K70, D71, S73, A74, D77, E78, T79, L80, D82, K83,T86, Y89, Q90, N93, D94, E96, A97, V99, 1100, Q101, G102, V103, G104,T106, E107, T108, P109, L110, M111, K112, E113, D114, L117, R120, K121,Q124, R125, T127, L128, K131, E132, K133, K134, Y135, S136, P137, C138,A145, M148, R149, S152, L153, N156, Q158, E159, S160, L161, R162, S163,K164 and E165, with position numbering relative to that of theinterferon-alpha 2a sequence identified herein as SEQ ID NO:32+R23K.

The following residues were determined to have on average more than 50%fractional ASA of their side chain: D2, L3, P4, Q5, T6, H7, S8, L9, R12,R13, M16, A19, S25, F27, S28, K31, R33, H34, D35, G37, E41, G44, N45,Q46, Q48, K49, N65, K70, A74, D77, E78, T79, D82, K83, T86, Y89, Q90,N93, D94, I100, Q101, G102, G104, T106, E107, T108, P109, L110, E113,D114, L117, R120, K121, Q124, R125, L128, K131, E132, K134, P137, R149,E159, L161, R162, S163, K164 and E165, with position numbering relativeto that of the interferon-alpha 2a sequence identified herein as SEQ IDNO:32+R23K.

Surface Exposure of Residues Corresponding to SEQ ID NO:1:

Owing to an insertion of an amino acid after position 44 of the humaninterferon-alpha 2 subtypes—such as, for example, interferon-alpha 2b(SEQ ID NO:32) and interferon-alpha 2a (SEQ ID NO:32+R23K)—in manyinterferon alpha sequences, including all of the known human interferonalpha sequences (apart from the IFN-alpha 2 subtypes) and certainpolypeptides of the invention, the position numbering of thesurface-exposed residues will be shifted by one residue past positionnumber 44 in, for example, the sequences shown in the alignment FIG. 2,relative to the numbering of the sequence denoted hIFNalpha 2b (SEQ IDNO:32).

Based on the above analysis, the following positions, numbered relativeto SEQ ID NO: 1, are considered to contain amino acid residues havingmore than 25% fractional ASA: positions 2, 3, 4, 5, 6, 7, 8, 9, 10, 12,13, 16, 19, 20, 22, 23, 24, 25, 26, 27, 28, 30, 31, 33, 34, 35, 37, 40,41, 42, 44, 46, 47, 49, 50, 51, 52, 59, 62, 63, 66, 69, 70, 71, 72, 74,75, 78, 79, 80, 81, 83, 84, 87, 90, 91, 94, 95, 97, 98, 100, 101, 102,103, 104, 105, 107, 108, 109, 110, 111, 112, 113, 114, 115, 118, 121,122, 125, 126, 128, 129, 132, 133, 134, 135, 136, 137, 138, 139, 146,149, 150, 153, 154, 157, 159, 160, 161, 162, 163, 164, 165, and 166.

Likewise, the following positions, again numbered relative to SEQ IDNO:1, are considered contain amino acid residues having on average morethan 50% fractional ASA of their side chain: 2, 3, 4, 5, 6, 7, 8, 9, 12,13, 16, 19, 25, 27, 28, 31, 33, 34, 35, 37, 41, 44, 46, 47, 49, 50, 66,71, 75, 78, 79, 80, 83, 84, 87, 90, 91, 94, 95, 101, 102, 103, 105, 107,108, 109, 110, 111, 114, 115, 118, 121, 122, 125, 126, 129, 132, 133,135, 138, 150, 160, 162, 163, 164, 165, and 166.

Example 2 Antiviral Activities of Interferon-Alpha Polypeptides

Patients with chronic HCV infection have initial viral loads in therange of 10⁴-10⁷ copies of HCV RNA/ml. Upon treatment with IFN-alpha,the viral load characteristically undergoes two distinct log-linearphases of decline reflecting two distinct mechanisms (FIG. 1B). Theinitial drop in viral load occurs in about the first two days and isbelieved to be due to the reduction in rate of virus production byinfected liver cells in the face of the IFN-alpha therapy.

The major technical challenge with HCV is that the virus cannot be grownin vitro and has only recently been cultured in tractable animal models.There are, however, viruses that replicate in vitro which are consideredto be useful surrogates for HCV viral replication. In vitro surrogateassays believed to be predictive of in vivo HCV antiviral activityinclude the assay described above, which measures the ability of testmolecules to protect cells from the cytopathic effect of viralinfection, using EMC RNA virus (EMCV) in the human liver-derived cellline HuH7.

Antiviral activities of some IFN-alpha polypeptides of the inventionwere assayed in the EMCV/HuH7 antiviral assay described in the Materialsand Methods section above. Some such polypeptides exhibited antiviralactivities about equal to or greater than that of a reference molecule,e.g., huIFN-alpha 2b (SEQ ID NO:32) or huIFN-alpha 2a (SEQ IDNO:32+R23K), as evidenced by the EC₅₀ (the concentration of sample whichyields half-maximal protective response in the assay) of the polypeptideof the invention being about equal to or less than the EC₅₀ of thereference molecule. Some such polypeptides of the invention exhibited atleast about a 1.5-fold higher, at least about a two-fold higher, atleast about a four-fold higher, at least about a five-fold higher, or atleast about a ten-fold higher antiviral activity than the referencemolecule (as evidenced by the EC₅₀ of the polypeptide being about 0.66×or lower, about 0.5× or lower, about 0.25× or lower, about 0.2× orlower, or about 0.1× or lower than the EC₅₀ of the reference molecule,respectively).

Table 7 below shows relative antiviral activities of exemplary IFN-alphapolypeptides of the invention, in comparison to IFN-alpha Con1 and humanIFN-alpha 2b assayed under the same conditions, expressed as antiviralactivity relative to huIFN-alpha 2b (EC₅₀ huIFN-alpha 2b/EC₅₀ sample).

TABLE 7 Antiviral Sample activity relative name Sequence to huIFNα-2bB9x14 SEQ ID NO:3 ≧10 B9x25 SEQ ID NO:12 ≧10 B9x16 SEQ ID NO:3 ≧10 +H47QB9x28 SEQ ID NO:12 ≧10 +E133K, A140S B9x23 SEQ ID NO:12 ≧10 +H47Q B9x18SEQ ID NO:3 ≧10 +H47Q, V51T, F55S, L56V, Y58H B9x22 SEQ ID NO:12 ≧10+V51T, F55S, L56V, Y58H B9x11 SEQ ID NO:3 ≧10 +E133K, A140S B9x17 SEQ IDNO:3 ≧10 +V51T, F55S, L56V, Y58H B9x27 SEQ ID NO:12 ≧10 +H47Q, E133K,A140S B9x12 SEQ ID NO:3 ≧10 +H47Q, E133K, A140S B9x21 SEQ ID NO:12 ≧10+H47Q, V51T, F55S, L56V, Y58H B9x26 SEQ ID NO:12 ≧5 +V51T, F55S, L56V,Y58H, E133K, A140S B9x24 SEQ ID NO:12 ≧5 +H47Q, V51T, F55S, L56V, Y58H,E133K, A140S B9x15 SEQ ID NO:3 ≧5 +H47Q, V51T, F55S, L56V, Y58H, E133K,A140S 25Ep05 SEQ ID NO:12 ≧2.5 +H47Q, V51T, F55S, L56V, Y58H, N72D,N95D, F154L, K160E, R161S, R164S IFNα-Con1 SEQ ID NO:43 ~2 huIFNα-2b SEQID NO:32 1

Example 3 T_(H)1 Differentiation Activities of Interferon-AlphaPolypeptides

Patients with chronic HCV infection have initial viral loads in therange of 10⁴-10⁷ copies of HCV RNA/ml. Upon treatment with IFN-alpha,the viral load characteristically undergoes two distinct log-linearphases of decline reflecting two distinct mechanisms (FIG. 1). Theinitial drop in viral load occurs in about the first two days and isbelieved to be due to the reduction in rate of virus production byinfected liver cells in response to IFN-alpha therapy. This reaches anew steady state after about two days at which time a second, lessrapid, log linear phase of viral clearance is observed. This secondphase is believed to be due to killing of infected liver cells byantigen specific T cells. IFN-alpha therapy is believed to play a keyrole in this biological response through the stimulation of antigenspecific T cells to differentiate into T_(H)1 cells.

Without being limited to a particular theory, it is proposed that aninterferon-alpha with an improved ability to stimulate differentiationof T_(H)0 cells to T_(H)1 cells may exhibit a more robust second phaseof viral clearance, and may therefore have improved efficacy in viralclearance. Based on this working hypothesis, an assay was developed tomeasure T_(H)1 differentiation activity of interferon-alphas on naïveT_(H)0 cells isolated from blood donors.

T_(H)1 differentiation activities of some IFN-alpha polypeptides of theinvention were assayed as described in the Materials and Methods sectionabove. Some such polypeptides of the invention exhibit T_(H)1differentiation activities about equal to that of a reference molecule,e.g., huIFN-alpha 2b (SEQ ID NO:32) or huIFN-alpha 2a (SEQ IDNO:32+R23K), as evidenced by the EC₅₀ (the concentration of sample whichyields half-maximal production of interferon-gamma in the assay) of thepolypeptide being about equal to the EC₅₀ of the reference molecule.Some polypeptides of the invention exhibited T_(H)1 differentiationactivities greater than that of the reference molecule, as evidenced bythe EC₅₀ of the polypeptide being lower than the EC₅₀ of the referencemolecule.

Example 4 Antiproliferative Activities of Interferon-Alpha Polypeptides

IFN-alpha inhibits proliferation of many cell types, although theantiproliferative effects often occur at higher doses than are requiredfor the antiviral response. Daudi cells are a human derivedEVB-transformed B cell line that is IFN-alpha sensitive. This IFN-alpharesponsive cell line serves as a useful probe of the antiproliferativeeffects of the IFN-alpha polypeptides of the invention. Furthermore,antiproliferative activity of IFN-alpha on megakaryocytes andneutrophils at high dose is believed to contribute to thrombocytopeniaand neutropenia, respectively. The Daudi antiproliferation assay mayserve as a useful surrogate assay for antiproliferative effects on theseother lymphoid cell types.

Antiproliferative activities of some IFN-alpha polypeptides of theinvention were assayed as described in the Materials and Methods sectionabove. Some polypeptides of the invention exhibit antiproliferativeactivities about equal to that of a reference molecule, such ashuIFN-alpha 2b (SEQ ID NO:32) or huIFN-alpha 2a (SEQ ID NO:32+R23K), asevidenced by the EC₅₀ (the concentration which yields half-maximalthymidine incorporation in the assay) of the polypeptide being aboutequal to the EC₅₀ of the reference molecule. Some polypeptides of theinvention exhibit antiproliferative activities about equal to or greaterthan that of the reference molecule, as evidenced by the EC₅₀ of thepolypeptide being about equal to or less than (e.g., about 0.75-fold,about 0.5-fold, or about 0.25-fold) the EC₅₀ of the reference molecule.Some polypeptides of the invention exhibit antiproliferative activitieswhich are about equal to or less than that of the reference molecule, asevidenced by the EC₅₀ of the polypeptide being about equal to or greaterthan the EC₅₀ of the reference molecule. Some such polypeptides of theinvention exhibit about a 0.75-fold or lower, about a 0.66-fold orlower, about a 0.5-fold or lower, about a 0.25-fold or lower, about a0.2-fold or lower, or about a 0.1-fold or lower antiproliferativeactivity than that of the reference molecule (as evidenced by the EC₅₀of the polypeptide being at least about 1.3-fold greater, at least about1.5-fold greater, at least about 2-fold greater, at least about 4-foldgreater, at least about 5-fold greater, or at least about 10-foldgreater than the EC₅₀ of the reference molecule, respectively); suchpolypeptides of the invention nevertheless exhibit a measurableantiproliferative activity.

Table 8 below shows relative antiproliferative activities of severalexemplary polypeptides of the invention, in comparison to humanIFN-alpha 2b and IFN-alpha Con1 assayed under the same conditions,expressed as antiproliferative activity relative to huIFN-alpha 2b (EC₅₀huIFN-alpha 2b/EC₅₀ sample).

TABLE 8 Antiproliferative Sample activity relative name Sequence tohuIFNα-2b B9x14 SEQ ID NO:3 ≦0.5 B9x25 SEQ ID NO:12 ≦0.5 B9x16 SEQ IDNO:3 ≦0.25 +H47Q B9x23 SEQ ID NO:12 ≦0.5 +H47Q B9x18 SEQ ID NO:3 ≦0.25+H47Q, V51T, F55S, L56V, Y58H B9x22 SEQ ID NO:12 ≦0.5 +V51T, F55S, L56V,Y58H B9x17 SEQ ID NO:3 ≦0.5 +V51T, F55S, L56V, Y58H B9x27 SEQ ID NO:12≦0.25 +H47Q, E133K, A140S B9x21 SEQ ID NO:12 ≦0.5 +H47Q, V51T, F55S,L56V, Y58H B9x26 SEQ ID NO:12 <0.25 +V51T, F55S, L56V, Y58H, E133K,A1405 B9x24 SEQ ID NO:12 ≦0.25 +H47Q, V51T, F55S, L56V, Y58H, E133K,A140S B9x15 SEQ ID NO:3 ≦0.25 +H47Q, V51T, F55S, L56V, Y58H, E133K,A140S IFNα-Con1 SEQ ID NO:43 ~1.5 huIFNα-2b SEQ ID NO:32 1

Example 5 PEGylation of Interferon-Alpha Polypeptides

Cys-PEGylation

A polypeptide of the invention which contains a free cysteine, (such as,for example, B9x14-CHO6 (SEQ ID NO:49), which contains a cysteine atposition 164), may be cysteine-PEGylated as follows. The polypeptide isfirst partially reduced with an equimolar concentration of TCEP(Triscarboxyethylphosphine) at 4° C. for 30 min in 50 mM MES, 100 mMNaCl, pH 6.0. The reduced polypeptide is then reacted with a 4 foldmolar excess of mPEG-MAL reagent (with a PEG moiety such as a 20 kDa or30 kDa linear mPEG, or a 40 kDa branched mPEG2) for 1 h at 4° C. underthe same conditions. The PEGylated reaction mixture is loaded on to aSP-Sepharose HP column equilibrated with 50 mM MES, pH 6.0, 100 mM NaCl.After a 10 CV (column volume) wash step a gradient from 0-600 mM NaCl isapplied to fractionate the PEGylated and unPEGylated fractions.Fractions are collected and aliquots are analyzed by SDS-PAGE. Fractionscontaining monoPEGylated species are pooled and formulated for assaysfor interferon-alpha activity as described above.

Lys-PEGylation

A polypeptide of the invention comprising the sequence SEQ ID NO:47 wasbuffer-exchanged into 50 mM sodium borate pH 9 by dialysis or gelfiltration, and was concentrated to between 1 and 5 mg/ml. The solutionwas cooled to 2-8° C. A 3- to 4-times molar excess of dry powderedNHS-mPEG2 40 kDa (Nektar; Huntsville, Ala.) over protein was added tothe protein solution and stirred using a stir bar. The stir speed waskept as high as possible without frothing. The reaction was completeafter about 1 h, after which the reaction was diluted 8-10 fold with 50mm sodium acetate, 100 mm NaCl pH 5 (optionally including 0.05%TWEEN80). The sample was filtered and loaded onto a HiTrap SPFF columnequilibrated with 50 mm sodium acetate, 100 mm NaCl pH 5. The column waswashed extensively with 10-15 column volumes of starting buffer. A 100mm to 1 M NaCl gradient in the same buffer was used to separatePEGylated proteins from non-PEGylated protein. Fractions containingPEGylated proteins were pooled and formulated for interferon-alphaactivity assays, and in some instances were further characterized byamino acid analysis and/or MALDI-TOF mass spectrometry. Preliminaryexperiments indicate that a polypeptide of the invention comprising thesequence SEQ ID NO:47, when lysine-PEGylated as described above,exhibits over 10-fold higher antiviral activity than a lysine-PEGylatedhuIFN-alpha 2a conjugate.

N-Terminal PEGylation

A polypeptide of the invention comprising the sequence SEQ ID NO:47 wasinitially buffer exchanged into 50 mM sodium borate pH 9 by dialysis orgel filtration, and concentrated to between 1 and 5 mg/ml, after whichthe polypeptide was buffer exchanged into 100 mM sodium phosphate pH 4by dialysis. A precipitate that on occasion formed during dialysisredissolved readily. The protein solution was cooled to 4° C. A 4 to 10times molar excess of dry powdered mPEG2-butylALD 40 kDa (Nektar;Huntsville, Ala.) over protein was added to the protein solution andstirred using a stir bar. The stir speed was kept as high as possiblewithout frothing. Subsequently a 1/10 vol of 200 mM NaCNBH3 in 100 mMpotassium phosphate pH 4 was added. The reaction was complete afterseveral hours but could be left overnight. The solution was diluted 5-10fold with 50 mM sodium acetate, 100 mM NaCl pH 4 (optionally including0.05% TWEEN80). The sample was filtered and loaded onto a HiTrap SPFFcolumn equilibrated with 50 mM sodium acetate, 100 mM NaCl pH 4. Thecolumn was washed extensively with 10-15 Column volumes of startingbuffer. A 100 mm to 1 M NaCl gradient in the same buffer was used toseparate PEGylated proteins from non-PEGylated protein. Fractionscontaining PEGylated protein were pooled and formulated forinterferon-alpha activity assays, and in some instances were furthercharacterized by amino acid analysis and/or MALDI-TOF mass spectrometry.Preliminary experiments indicate that a polypeptide of the inventioncomprising the sequence SEQ ID NO:47, when N-terminally PEGylated asdescribed above, exhibits over 10-fold higher antiviral activity than alysine-PEGylated huIFN-alpha 2a conjugate.

Example 6 In Vivo Assays

Measurement of Half-Life of a Polypeptide or Conjugate of the Invention

Measurement of biological or serum half-life may be carried out in anumber of ways described in the literature. For example, biologicalhalf-life may be determined using an ELISA method to detect serum levelsof interferon-alpha after e.g. subcutaneous or intramuscularadministration. Use of an ELISA method to determine the pharmacokineticsof interferon-alpha administered subcutaneously is e.g. described byRostaing et al. (1998), J. Am. Soc. Nephrol. 9(12): 2344-48. Merimsky etal. (1991), Cancer Chemother. Pharmacol. 27(5); 406-8, describe thedetermination of the serum level of an interferon-alpha administeredintramuscularly.

Determining In Vitro Immunogenicity

Reduced immunogenicity of a polypeptide or conjugate of the inventioncan be determined by use of an ELISA method measuring theimmunoreactivity of the molecule relative to a reference molecule orpreparation, typically a known interferon-alpha protein. The ELISAmethod is based on antibodies from patients treated with the referenceprotein. The immunogenicity is considered to be reduced when thepolypeptide or conjugate of the invention has a statisticallysignificant lower response in the assay than the reference molecule orpreparation.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and scope of the appended claims. For example, all thetechniques and apparatus described above may be used in variouscombinations. All publications, patents, patent applications, and/orother documents cited in this application are incorporated herein byreference in their entirety for all purposes to the same extent as ifeach individual publication, patent, patent application, and/or otherdocument were individually indicated to be incorporated herein byreference in its entirety for all purposes.

1. A method for reducing the level of a virus in the serum of a patient infected with the virus, comprising administering to the patient a polypeptide in an amount effective to reduce the level of virus in the serum compared to the level present prior to the start of treatment, wherein the polypeptide comprises a sequence which differs in 0 to 8 amino acid positions from the sequence of SEQ ID NO:12 or SEQ ID NO:10 and wherein the polypeptide exhibits antiviral activity.
 2. The method of claim 1 wherein the virus is Hepatitis C Virus.
 3. The method of claim 1 wherein the virus is Hepatitis B Virus.
 4. A method for reducing the level of a virus in the serum of a patient infected with the virus, comprising administering to the patient a conjugate in an amount effective to reduce the level of virus in the serum compared to the level present prior to the start of treatment, wherein the conjugate comprises: a) a polypeptide comprising a sequence which differs in 0 to 8 amino acid positions from the sequence of SEQ ID NO:12 or SEQ ID NO:10; and b) a non-polypeptide moiety covalently attached to the polypeptide, and wherein the conjugate exhibits antiviral activity.
 5. The method of claim 4 wherein the virus is Hepatitis C Virus.
 6. The method of claim 4 wherein the virus is Hepatitis B Virus.
 7. The method of claim 4 wherein the non-polypeptide moiety is a polymer.
 8. The method of claim 7 wherein the polymer is polyethylene glycol.
 9. The method of claim 8 wherein a polyethylene glycol moiety is covalently attached to a cysteine residue.
 10. The method of claim 8 wherein a polyethylene glycol moiety is covalently attached to a lysine residue or to the N-terminal amino group.
 11. The method of claim 8 wherein a polyethylene glycol moiety is covalently attached to a lysine residue.
 12. The method of claim 8 wherein a polyethylene glycol moiety is covalently attached to the N-terminal amino group.
 13. The method of claim 8, wherein at least two polyethylene glycol moieties are attached to the polypeptide and wherein each polyethylene glycol moiety is covalently attached to a different amino acid residue of the polypeptide.
 14. The method of claim 13, wherein the at least two polyethylene glycol moieties are attached to different lysine residues.
 15. The method of claim 13, wherein one of the at least two polyethylene glycol moieties is attached to the N-terminal amino group and one of the at least two polyethylene glycol moieties is attached to a lysine residue.
 16. The method of claim 4 wherein the non-polypeptide moiety is a sugar.
 17. The method of claim 16 wherein the sugar is attached to an N-glycosylation site.
 18. A method of treating a patient infected with Hepatitis C Virus comprising administering to the patient a pharmaceutically acceptable excipient and a therapeutically effective amount of a conjugate, wherein the conjugate comprises: a) a polypeptide comprising a sequence which differs in 0 to 8 amino acid positions from the sequence of SEQ ID NO:12 or SEQ ID NO:10; and b) a non-polypeptide moiety covalently attached to the polypeptide, and wherein the conjugate exhibits antiviral activity.
 19. The method of claim 18 wherein the conjugate is administered by weekly subcutaneous injection.
 20. The method of claim 18 wherein the conjugate is administered by subcutaneous injection every two weeks.
 21. The method of claim 18 wherein the non-polypeptide moiety is a polymer.
 22. The method of claim 21 wherein the polymer is polyethylene glycol.
 23. The method of claim 22 wherein a polyethylene glycol moiety is covalently attached to a cysteine residue.
 24. The method of claim 22 wherein a polyethylene glycol moiety is covalently attached to a lysine residue or to the N-terminal amino group.
 25. The method of claim 22 wherein a polyethylene glycol moiety is covalently attached to a lysine residue.
 26. The method of claim 22 wherein a polyethylene glycol moiety is covalently attached to the N-terminal amino group.
 27. The method of claim 22, wherein at least two polyethylene glycol moieties are attached to the polypeptide and wherein each polyethylene glycol moiety is covalently attached to a different amino acid residue of the polypeptide.
 28. The method of claim 27, wherein the at least two polyethylene glycol moieties are attached to different lysine residues.
 29. The method of claim 27, wherein one of the at least two polyethylene glycol moieties is attached to the N-terminal amino group and one of the at least two polyethylene glycol moieties is attached to a lysine residue.
 30. The method of claim 18 wherein the non-polypeptide moiety is a sugar.
 31. The method of claim 30 wherein the sugar is attached to an N-glycosylation site. 